Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus.

The BsgA protease of Myxococcus xanthus is an intracellular protease closely related to the Lon protease of Escherichia coli. BsgA is required for normal levels of developmentally induced gene expression. In this report, we describe the identification of mutations that suppress the developmental defect of bsgA mutants. These mutations localized to the spdR gene (suppressor protease deficiency regulator) that appears to play a role in the regulation of early developmental gene expression. Mutations in spdR fully restored the ability of a bsgA mutant to form fruiting bodies and spores and, with one exception, restored the expression of several development-specific lacZ fusions. spdR mutants exhibited characteristic phenotypic properties including increased expression of the development-specific tps gene during vegetative growth, formation of fruiting bodies and spores on semi-rich nutrient medium and completion of starvation-induced development in a shorter time period than wild-type strains. The spdR locus was cloned and sequenced and found to encode a member of the NtrC family of two-component transcriptional regulators. One interpretation of these data is that SpdR acts, directly or otherwise, to regulate developmental gene expression negatively and that the BsgA protease is required to relieve this inhibitory effect at the onset of development. However, Western immunoblot analysis indicated that SpdR is present at a relatively constant level during growth and early development in both wild-type and BsgA protease-deficient cells. This finding suggests that BsgA does not function to degrade SpdR at the onset of development.

Biochemical and molecular analysis of phospholipase C and phospholipase D activity in mycobacteria.

Resurgence of mycobacterial infections in the United States has led to an intense effort to identify potential virulence determinants in the genus Mycobacterium, particularly ones that would be associated with the more virulent species (e.g., Mycobacterium tuberculosis). Thin-layer chromatography (TLC) using radiolabeled phosphatidylcholine and sphingomyelin as substrates indicated that cell extracts of M. tuberculosis contain both phospholipase C (PLC) and phospholipase D (PLD) activities. In contrast, only PLD activity was detected in cell extracts of M. smegmatis. Neither activity was detected in cell-free culture supernatants from these organisms. We and others recently identified two open reading frames in M. tuberculosis with the potential to encode proteins which are highly homologous to the nonhemolytic (PlcN) and hemolytic (PlcH) phospholipase C enzymes of Pseudomonas aeruginosa. In contrast to the plc genes in P. aeruginosa, which are considerably distal to each other (min 34 and 64 on the chromosome), the mycobacterial genes, designated mpcA and mpcB, are tandemly arranged in the same relative orientation and separated by only 191 bp. Both the mpcA and the mpcB genes were individually cloned in M. smegmatis, and PLC activity was expressed from each gene in this organism. Hybridization experiments using the mpcA and the mpcB genes as probes under conditions of moderate stringency identified sequences homologous to these genes in M. bovis, M. bovis BCG, and M. marinum but not in several other Mycobacterium species, including M. smegmatis, M. avium, and M. intracellulare. TLC analysis using radiolabeled substrates indicated that M. bovis and M. marinum cell extracts contain PLC and PLD activities, but only PLD activity was detected in M. bovis BCG cell extracts. Sphingomyelinase activity was also detected in whole-cell extracts of M. tuberculosis, M. marinum, M. bovis, and M. bovis BCG, but this activity was not detected in extracts of M. smegmatis. Sphingomyelinase activity was detected in cell extracts from M. smegmatis harboring either recombinant mpcA or mpcB. These data indicate that PLC and sphingomyelinase activities are associated with the most virulent mycobacterial species, while PLD activity was detected in both virulent and saprophytic strains.

Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.

omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403. We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or beta-galactosidase accumulation in cells containing Tn5 lac omega 4403. The putative transcriptional start site was mapped, and sequences centered near -10 and -35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli sigma70 RNA polymerase. However, deletions showed that an essential promoter element lies between -80 and -72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression.

Using a phase-locked mutant of Myxococcus xanthus to study the role of phase variation in development.

The bacterium Myxococcus xanthus undergoes a primitive developmental cycle in response to nutrient deprivation. The cells aggregate to form fruiting bodies in which a portion of the cells differentiate into environmentally resistant myxospores. During the growth portion of the M. xanthus life cycle, the organism also undergoes a phase variation, in which cells alternate between yellow and tan colony-forming variants. Phase variation occurs in our laboratory strain (M102, a derivative of DK1622) at a frequency high enough that a single colony of either the yellow or the tan phase already contains cells of the alternate phase. In this study we demonstrate that tan cells within a predominantly yellow population of phase variation-proficient cells are preferentially recovered as heat- and sonication-resistant spores. To further investigate the possibility of a differential role of tan and yellow cells during development, a tan-phase-locked mutant was used to compare the developmental phenotypes of a pure tan population with a predominantly yellow, phase variation-proficient population. Pure tan-phase populations did not produce fruiting bodies or mature spores under conditions in which predominantly yellow wild-type populations did so efficiently. Pure populations of tan-phase cells responded to developmental induction by changing from vegetative rod-shaped cells to round forms but were unable to complete the maturation to heat- and sonication-resistant, refractile spores. The developmental defect of a tan-phase-locked mutant was rescued by the addition of phase variation-proficient cells from a predominantly yellow culture. In such mixtures the tan-phase-locked mutant not only completed the process of forming spores but also was again preferentially represented among the viable spores. These findings suggest the intriguing possibility that the tan-phase cells within the vegetative population entering development are the progenitors of spores and implicate a requirement for yellow-phase cells in spore maturation.

Use of a phase variation-specific promoter of Myxococcus xanthus in a strategy for isolating a phase-locked mutant.

The bacterium Myxococcus xanthus alternates between two colony types distinguished by colony morphology and pigmentation. Because the two phases are interconvertible, this phenomenon has been termed phase variation. In one phase, the colonies are bright yellow, rough, and swarming. In the alternate phase, the colonies are tan and mucoid with smooth edges. During exponential vegetative growth, the populations within a colony reach an equilibrium of approximately 99% yellow and 1% tan cells. Neither the biological function nor the genetic mechanism of phase variation is currently understood. To investigate phase variation, a yellow-phase-specific promoter was identified by Tn5lac mutagenesis. A tan-phase-locked mutant was isolated by a strategy, described in this study, which involved introducing a selectable marker expressed under phase-regulated expression. This was accomplished by a fusion of the cloned yellow-phase-specific promoter to a promoterless kanamycin resistance gene. The defect in the phase-locked mutant, given the designation var-683, caused the rate of switching from the tan to yellow phase to be reduced by at least 10(3)-fold below the wild-type rate of switching. This strain will provide a stable tan population for genetic and biological analysis. Evidence is presented for the existence of a transcriptional regulator which controls the expression of phase-regulated promoters.

Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein-ubiquinone oxidoreductase.

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) in the inner mitochondrial membrane accepts electrons from electron-transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64-kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF-QO and used these as probes to clone a full-length human ETF-QO cDNA. The deduced human ETF-QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin-like iron-sulfur clusters. A cleavable 33-amino-acid sequence is also predicted at the amino terminus of the 67-kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67-kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF-QO is synthesized as a 67-kDa precursor which is targeted to mitochondria and processed in a single step to a 64-kDa mature form located in the mitochondrial membrane. The detergent-solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron-sulfur cluster are properly inserted into the heterologously expressed protein.

Cloning, sequencing, and expression of the genes encoding subunits of Paracoccus denitrificans electron transfer flavoprotein.

The genes encoding the two subunits of Paracoccus denitrificans electron transfer flavoprotein (ETF) were identified by screening a genomic library constructed in pBluescript II SK+ with probes generated by amplification of genomic sequences by the polymerase chain reaction. Primers for the polymerase chain reaction were designed based on peptide sequences from purified Paracoccus ETF subunits. The genes are arranged in tandem in the genomic DNA with the deoxyadenylic acid residue in the TGA termination codon of the small subunit providing the deoxyadenylic acid residue for the ATG initiating codon of the large subunit. The deduced amino acid sequences of the ETF subunits exhibits extensive sequence identity with the human ETF subunits. The Paracoccus ETF is expressed from the pBluescript vector in Escherichia coli, yielding 30 mg of purified, catalytically active protein per liter of culture.

Myxococcus xanthus encodes an ATP-dependent protease which is required for developmental gene transcription and intercellular signaling.

The bsgA gene of Myxococcus xanthus plays an essential role in the regulation of early gene expression during fruiting body formation and sporulation. bsgA mutants behave as though unable to initiate a required cell-cell interaction and consequently fail to transcribe normal levels of many developmentally induced genes. We determined the nucleotide sequence of bsgA, which predicts a single gene encoding a 90.4-kDa protein. The deduced BsgA protein shares 45 and 48% amino acid identity with the lon genes of Escherichia coli and Bacillus brevis, respectively. The cloned bsgA gene was expressed in E. coli, and the BsgA protein was partially purified and found, like its E. coli homolog, to be an ATP-dependent protease. Thus, the basis for the phenotype of bsgA mutants is likely to be a defect in intracellular proteolysis.

An 87-kilodalton glucan-binding protein of Streptococcus sobrinus B13.

An 87-kDa glucan-binding protein (GBP) of Streptococcus sobrinus B13 (serotype d) was isolated and purified from extracellular culture supernatant by using affinity chromatography on Sephadex G-50 and elution with a guanidine HCl gradient. Western blot (immunoblot) analysis showed it to be antigenically related, but not completely identical, to the 74-kDa GBP of Streptococcus mutans Ingbritt. The 87-kDa GBP has no glucosyltransferase activity. A possible role for this GBP in the cariogenicity of S. sobrinus B13 is suggested.

Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus.

Developmental mutants of Myxococcus xanthus have been previously described which appear to be defective in required cell-cell interactions. These mutants fall into four phenotypic classes, Asg, Bsg, Csg, and Dsg, each of which is unable to differentiate into spores but can be rescued by extracellular complementation by wild-type cells or by mutants of a different class. We report the identification of one of the loci in which mutations result in a Bsg phenotype. The cloned locus was contained on a 12-kilobase EcoRI fragment and then localized by subcloning and a combination of in vitro and transposon mutagenesis. All mutations in this locus behave as a single complementation group, which we designate bsgA (formerly ssbA). Each of the bsgA mutations results in a nonsporulating phenotype, which can be rescued by extracellular complementation. Furthermore, we report that the bsgA mutants have a distinctive interaction with wild-type cells when vegetatively growing, swarming colonies converge.

Identification and characterization of the Myxococcus xanthus bsgA gene product.

The bsgA mutants of Myxococcus xanthus are blocked at a very early stage of the developmental program. They fail to produce fruiting bodies or to sporulate under normal conditions but can be rescued by extracellular complementation in mixtures with wild-type cells. A bsgA-lacZ gene fusion was constructed and expressed in Escherichia coli. The resulting fusion protein, which has beta-galactosidase enzyme activity, was partially purified by affinity chromatography and preparative polyacrylamide gel electrophoresis. The protein was used to immunize mice, which produced a hybridoma secreting monoclonal antibody that was specific for the bsgA gene product. The monoclonal antibody was used in Western blot (immunoblot) experiments to determine the apparent cellular location of the bsgA protein in M. xanthus and to compare the level of this protein at various times in the Myxococcus life cycle.

Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus.

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.

Developmental cell interactions in Myxococcus xanthus and the spoC locus.

The product(s) of the Myxococcus xanthus spoC locus is required for two multicellular activities in fruiting body development, rippling and sporulation. Ripples, which are formed early in development, are spatially separated ridges of cells that move synchronously. Myxospores are heat-resistant resting cells that are formed near the end of the developmental process. To investigate the function of spoC, it was cloned in an Escherichia coli plasmid, then transferred to M. xanthus by specialized transduction with coliphage P1. The plasmid, which cannot replicate in M. xanthus, integrated into the M. xanthus chromosome, producing two copies of the spoC locus in tandem. Cells containing one copy of a mutant allele and one copy of the wild-type allele displayed the wild-type phenotype. Cells containing two different mutant alleles failed to ripple or sporulate, implying that all four independent spoC mutations are in the same gene or unit of transcription. Homozygous mutant duplications arose from constructions in which DNA from a spo(+) donor was transduced into a spoC recipient, or vice versa, at an average frequency of 14%, indicating that gene conversion was a frequent event.

Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.

The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation.

Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate.

Isolates of Escherichia coli from human urinary tract infections frequently express adherence properties found less often among normal intestinal isolates. These properties include adherence to human uroepithelial cells and primary monkey kidney cells, as well as D-mannose-resistant hemagglutination of human erythrocytes, and they are mediated by a pilus type different from type 1. The genes encoding this pilus type (pyelonephritis-associated pili, pap) and those encoding type 1 pili have been cloned from a urinary tract infection isolate of E. coli and transferred to an E. coli K-12 derivative. The recombinant plasmids were found to express functional pili and to endow the new host with all of the adherence properties of the urinary tract infection isolate. Both pilus types were found to be genetically distinct, and unlike the adherence genes from bovine, porcine, and human diarrheal isolates, both were found to be chromosomally encoded.

Identification of the protein encoded by the transposable element Tn3 which is required for its transposition.

Protein products have now been identified which account for the entire coding capacity of the transposable element Tn3. Mutations in Tn3 have allowed us to map the genes encoding each of these peptides and to identify their role in transposition. We have found that only a single Tn3-encoded peptide is required for transposition. Expression of this peptide is repressed by the product of a second gene, which is itself autogenously regulated.