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Characterization of bcsA mutations that bypass two distinct signaling requirements for Myxococcus xanthus development.

The BsgA protease is required for starvation-induced development in Myxococcus xanthus. Bypass suppressors of a bsgA mutant were isolated to identify genes that may encode additional components of BsgA protease-dependent regulation of development. Strain M951 was isolated following Tn5 mutagenesis of a bsgA mutant and was capable of forming fruiting bodies and viable spores in the absence of the BsgA protease. The Tn5Omega951 insertion was localized to a gene, bcsA, that encodes a protein that has significant amino acid similarity to a group of recently described flavin-containing monooxygenases involved in styrene catabolism. Mutations in bcsA bypassed the developmental requirements for both extracellular B and C signaling but did not bypass the requirement for A signaling. Bypass of the B-signaling requirement by the bcsA mutation was accompanied by restored expression of a subset of developmentally induced lacZ fusions to the BsgA protease-deficient strain. bcsA mutant cells developed considerably faster than wild-type cells at low cell density and altered transcriptional levels of a developmentally induced, cell-density-regulated gene (Omega4427), suggesting that the bcsA gene product may normally act to inhibit development in a cell-density-regulated fashion. Bypass of the requirements for both B and C signaling by bcsA mutations suggests a possible link between these two genetically, biochemically, and temporally distinct signaling requirements.

Amino Acid Sequence↗

A CheW homologue is required for Myxococcus xanthus fruiting body development, social gliding motility, and fibril biogenesis.

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.

Agglutination↗

Analysis of dofA, a fruA-dependent developmental gene, and its homologue, dofB, in Myxococcus xanthus.

The developmentally regulated gene dofA, identified from pulse-labeling experiments by two-dimensional gel electrophoresis, and its homologue, dofB, were cloned and characterized in Myxococcus xanthus. Deletion of dofA and dofB did not affect the vegetative growth and development of M. xanthus. dofA was specifically expressed during development, while dofB expression was observed during vegetative growth and development. The dofA-lacZ fusion was introduced into a fruA mutant and A, B, C, D, and E extracellular signal mutants. The pattern of dofA expression in the C signal mutant was similar to that of the wild-type strain, while dofA expression was not detected in the fruA mutant. These results are consistent with those of the pulse-labeling experiments. dofA expression was reduced in A and E signal mutants, whereas dofA expression was delayed in B and D signal mutants. The patterns of expression of the dofA gene in the fruA mutant and the five signal mutants are strikingly similar to that of the tps gene, which encodes protein S, a major component of the outer surface of the myxospore; this result suggests that the dofA and tps genes are similarly regulated. The involvement of a highly GC-rich inverted repeat sequence (underlined), CGGCCCCCGATTCGTCGGGGGCCG, in developmentally regulated dofA expression is suggested.

Amino Acid Sequence↗

Bypass of A- and B-signaling requirements for Myxococcus xanthus development by mutations in spdR.

Mutations in spdR, previously reported to bypass the developmental requirement for B-signaling in Myxococcus xanthus, also bypass the requirement for A-signaling but not C-, D-, or E-signaling. Mutations in spdR restored nearly wild-type levels of sporulation to representative A-signal-deficient mutants carrying asgA476, asgB480, and asgC767 and improved the quality of fruiting body formation in the asgB480 mutant. The defect in A-factor production by the asgB480 mutant was not restored in the spdR2134 asgB480 double mutant.

4-Butyrolactone↗

Mapping of Myxococcus xanthus social motility dsp mutations to the dif genes.

Myxococcus xanthus dsp and dif mutants have similar phenotypes in that they are deficient in social motility and fruiting body development. We compared the two loci by genetic mapping, complementation with a cosmid clone, DNA sequencing, and gene disruption and found that 16 of the 18 dsp alleles map to the dif genes. Another dsp allele contains a mutation in the sglK gene. About 36.6 kb around the dsp-dif locus was sequenced and annotated, and 50% of the genes are novel.

Bacterial Proteins↗

The DevT protein stimulates synthesis of FruA, a signal transduction protein required for fruiting body morphogenesis in Myxococcus xanthus.

Fruiting body formation in Myxococcus xanthus involves three morphologic stages---rippling, aggregation, and sporulation---all of which are induced by the cell surface-associated C-signal. We analyzed the function of the DevT protein, a novel component in the C-signal response pathway. A mutant carrying an in-frame deletion in the devT gene displays delayed aggregation and a cell autonomous sporulation defect, whereas it remains rippling proficient. To further define the function of DevT, the methylation pattern of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein homologue, was examined in the Delta devT mutant, and we found that DevT is required for methylation of FrzCD during development. Specifically, DevT was found to be required for the C-signal-dependent methylation of FrzCD. The Delta devT mutant produced wild-type levels of C-signal. However, accumulation of the FruA response regulator protein, which is essential for the execution of the C-signal-dependent responses, was reduced in the Delta devT mutant. The DevT protein was found to stimulate the developmentally activated transcription of the fruA gene. Epistasis analyses indicate that DevT acts independently of the A- and E-signals to stimulate fruA transcription. These findings suggest that the developmental defects of the Delta devT mutant are associated with a lack of FruA to ensure a proper response to the C-signal during the aggregation and sporulation stages of development.

Bacterial Proteins↗

An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus.

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.

Amino Acid Sequence↗

Role for vitamin B(12) in light induction of gene expression in the bacterium Myxococcus xanthus.

A light-inducible promoter (P(B)) drives the carB operon (carotenoid genes) of the bacterium Myxococcus xanthus. A gene encoding a regulator of carotenoid biosynthesis was identified by studying mutant strains carrying a transcriptional fusion to P(B) and deletions in three candidate genes. Our results prove that the identified gene, named carA, codes for a repressor of the P(B) promoter in the dark. They also show that the carA gene product does not participate in the light activation of two other promoters connected with carotenoid synthesis or its regulation in M. xanthus. CarA is a novel protein consisting of a DNA-binding domain of the family of MerR helix-turn-helix transcriptional regulators, directly joined to a cobalamin-binding domain. In support of this, we report here that the presence of vitamin B(12) or some other cobalamin derivatives is absolutely required for activation of the P(B) promoter by light.

Amino Acid Sequence↗

Novel developmental genes, fruCD, of Myxococcus xanthus: involvement of a cell division protein in multicellular development.

Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, two novel developmental genes, fruC and fruD, of M. xanthus were identified and characterized. The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including Bacillus subtilis. Vegetative cells of the fruD mutant exhibited a filamentous phenotype. The fruC and fruD mutants displayed similar delayed-development phenotypes. The formation of tightly aggregated mounds by fruC and fruD mutants was slower than that by the wild-type strain. Spore formation by the fruC and fruD mutants initiated after 30 h poststarvation, whereas wild-type M. xanthus initiated spore formation after 18 h. The fruCD genes were constitutively expressed as an operon during vegetative growth and development. S1 mapping revealed that transcription initiation sites of the fruCD operon were located 114 (P1) and 55 bp (P2) upstream of the fruC initiation codon. Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development. The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.

Amino Acid Sequence↗

The Stigmatella aurantiaca homolog of Myxococcus xanthus high-mobility-group A-type transcription factor CarD: insights into the functional modules of CarD and their distribution in bacteria.

Transcriptional factor CarD is the only reported prokaryotic analog of eukaryotic high-mobility-group A (HMGA) proteins, in that it has contiguous acidic and AT hook DNA-binding segments and multifunctional roles in Myxococcus xanthus carotenogenesis and fruiting body formation. HMGA proteins are small, randomly structured, nonhistone, nuclear architectural factors that remodel DNA and chromatin structure. Here we report on a second AT hook protein, CarD(Sa), that is very similar to CarD and that occurs in the bacterium Stigmatella aurantiaca. CarD(Sa) has a C-terminal HMGA-like domain with three AT hooks and a highly acidic adjacent region with one predicted casein kinase II (CKII) phosphorylation site, compared to the four AT hooks and five CKII sites in CarD. Both proteins have a nearly identical 180-residue N-terminal segment that is absent in HMGA proteins. In vitro, CarD(Sa) exhibits the specific minor-groove binding to appropriately spaced AT-rich DNA that is characteristic of CarD or HMGA proteins, and it is also phosphorylated by CKII. In vivo, CarD(Sa) or a variant without the single CKII phosphorylation site can replace CarD in M. xanthus carotenogenesis and fruiting body formation. These two cellular processes absolutely require that the highly conserved N-terminal domain be present. Thus, three AT hooks are sufficient, the N-terminal domain is essential, and phosphorylation in the acidic region by a CKII-type kinase can be dispensed with for CarD function in M. xanthus carotenogenesis and fruiting body development. Whereas a number of hypothetical proteins homologous to the N-terminal region occur in a diverse array of bacterial species, eukaryotic HMGA-type domains appear to be confined primarily to myxobacteria.

AT-Hook Motifs↗

Membrane localization of motility, signaling, and polyketide synthetase proteins in Myxococcus xanthus.

Myxococcus xanthus cells coordinate cellular motility, biofilm formation, and development through the use of cell signaling pathways. In an effort to understand the mechanisms underlying these processes, the inner membrane (IM) and outer membrane (OM) of strain DK1622 were fractionated to examine protein localization. Membranes were enriched from spheroplasts of vegetative cells and then separated into three peaks on a three-step sucrose gradient. The high-density fraction corresponded to the putative IM, the medium-density fraction corresponded to a putative hybrid membrane (HM), and the low-density fraction corresponded to the putative OM. Each fraction was subjected to further separation on discontinuous sucrose gradients, which resulted in discrete protein peaks for each major fraction. The purity and origin of each peak were assessed by using succinate dehydrogenase (SDH) activity as the IM marker and reactivities to lipopolysaccharide core and O-antigen monoclonal antibodies as the OM markers. As previously reported, the OM markers localized to the low-density membrane fractions, while SDH localized to high-density fractions. Immunoblotting was used to localize important motility and signaling proteins within the protein peaks. CsgA, the C-signal-producing protein, and FibA, a fibril-associated protease, were localized in the IM (density, 1.17 to 1.24 g cm(-3)). Tgl and Cgl lipoproteins were localized in the OM, which contained areas of high buoyant density (1.21 to 1.24 g cm(-3)) and low buoyant density (1.169 to 1.171 g cm(-3)). FrzCD, a methyl-accepting chemotaxis protein, was predominantly located in the IM, although smaller amounts were found in the OM. The HM peaks showed twofold enrichment for the type IV pilin protein PilA, suggesting that this fraction contained cell poles. Two-dimensional polyacrylamide gel electrophoresis revealed the presence of proteins that were unique to the IM and OM. Characterization of proteins in an unusually low-density membrane peak (1.072 to 1.094 g cm(-3)) showed the presence of Ta-1 polyketide synthetase, which synthesizes the antibiotic myxovirescin A.

Anti-Bacterial Agents↗

TodK, a putative histidine protein kinase, regulates timing of fruiting body morphogenesis in Myxococcus xanthus.

In response to starvation, Myxococcus xanthus initiates a developmental program that results in the formation of spore-filled multicellular fruiting bodies. Fruiting body formation depends on the temporal and spatial coordination of aggregation and sporulation. These two processes are induced by the cell surface-associated C signal, with aggregation being induced after 6 h and sporulation being induced once cells have completed the aggregation process. We report the identification of TodK, a putative histidine protein kinase of two-component regulatory systems that is important for the correct timing of aggregation and sporulation. Loss of TodK function results in early aggregation and early, as well as increased levels of, sporulation. Transcription of todK decreases 10-fold in response to starvation independently of the stringent response. Loss of TodK function specifically results in increased expression of a subset of C-signal-dependent genes. Accelerated development in a todK mutant depends on the known components in the C-signal transduction pathway. TodK is not important for synthesis of the C signal. From these results we suggest that TodK is part of a signal transduction system which converges on the C-signal transduction pathway to negatively regulate aggregation, sporulation, and the expression of a subset of C-signal-dependent genes. TodK and the SdeK histidine protein kinase, which is part of a signal transduction system that converges on the C-signal transduction pathway to stimulate aggregation, sporulation, and C-signal-dependent gene expression, act in independent genetic pathways. We suggest that the signal transduction pathways defined by TodK and SdeK act in concert with the C-signal transduction pathway to control the timing of aggregation and sporulation.

Amino Acid Sequence↗

Global mutational analysis of NtrC-like activators in Myxococcus xanthus: identifying activator mutants defective for motility and fruiting body development.

The multicellular developmental cycle of Myxococcus xanthus requires large-scale changes in gene transcription, and recent findings indicate that NtrC-like activators play a prominent role in regulating these changes. In this study, we made insertions in 28 uncharacterized ntrC-like activator (nla) genes and found that eight of these insertions cause developmental defects. Hence, these results are consistent with the idea that M. xanthus uses a series of different NtrC-like activators during fruiting body development. Four of the eight developmental mutants we identified have motility defects. The nla1, nla19, and nla23 mutants show S-motility defects, while the nla24 mutant shows defects in both S-motility and A-motility. During development, aggregation of the nla1, nla19, and nla23 mutants is delayed slightly and the nla24 mutant shows no signs of aggregation or sporulation. The nla4, nla6, nla18, and nla28 mutants have no appreciable loss in motility, but they fail to aggregate and to sporulate normally. The nla18 mutant belongs to a special class of developmental mutants whose defects can be rescued when they are codeveloped with wild-type cells, suggesting that nla18 fails to produce a cell-cell signal required for development. The three remaining activator mutants, nla4, nla6, and nla28, appear to have complex developmental phenotypes that include deficiencies in cell-cell developmental signals.

Bacterial Proteins↗

Characterization of the integrase gene and attachment site for the Myxococcus xanthus bacteriophage Mx9.

Bacteriophage Mx9 is a temperate phage that infects Myxococcus xanthus. It lysogenizes the bacteria by integrating into the bacterial chromosome by site-specific recombination at one of two sites, attB1 or attB2. Integration at attB1 results in deletion of DNA between the two attB sites. The attB2 site lies within the 5' region of the M. xanthus tRNA(Gly) gene. Mx9 integration requires a single protein, Int. Analysis of integration revealed that the phage attachment site (attP) is contained in the int gene and that upon integration, the 3' end of the int gene is altered. Plasmids containing fusions of the pilA or mgl promoter to lacZ integrated at either Mx9 attB site have higher levels of transcription than the same fusions integrated at the Mx8 attB site.

3' Flanking Region↗

Role of two novel two-component regulatory systems in development and phosphatase expression in Myxococcus xanthus.

We have cloned a two-component regulatory system (phoR2-phoP2) of Myxococcus xanthus while searching for genes that encode proteins with phosphatase activity, where phoR2 encodes the histidine kinase and phoP2 encodes the response regulator. A second system, phoR3-phoP3, was identified and isolated by using phoP2 as a probe. These two systems are quite similar, sharing identities along the full-length proteins of 52% on the histidine kinases and 64% on the response regulators. The predicted structures of both kinases suggest that they are anchored to the membrane, with the sensor domains being located in the periplasmic space and the kinase domains in the cytoplasm. The response regulators (PhoP2 and PhoP3) exhibit a helix-loop-helix motif typical of DNA-binding proteins in the effector domains located in the C-terminal region. Studies on two single-deletion mutants and one double-deletion mutant have revealed that these systems are involved in development. Mutant fruiting bodies are not well packed, originating loose and flat aggregates where some myxospores do not reshape properly, and they remain as elongated cells. These systems are also involved in the expression of Mg-independent acid and neutral phosphatases, which are expressed during development. The neutral phosphatase gene is especially dependent on PhoP3. Neither PhoP2 nor PhoP3 regulates the expression of alkaline phosphatases and the pph1 gene.

Amino Acid Sequence↗

cis Elements necessary for developmental expression of a Myxococcus xanthus gene that depends on C signaling.

Cell contact-mediated C signaling coordinates morphogenesis and gene expression during development of Myxococcus xanthus. One promoter that depends on C signaling for transcription lies upstream of Omega4403, the site of a Tn5 lac insertion in the genome. The Omega4403 promoter has a C-box sequence centered at -49 bp that matches the consensus 5'-CAYYCCY-3', which is found in several C-signal-dependent promoters. Mutational analysis of the Omega4403 promoter region was performed to test the importance of the C box and to identify other cis-acting elements. A 6-bp change in the -10 region eliminated promoter activity, but a 6-bp change in the -35 region decreased activity only about twofold. Certain single-base-pair changes in the C box centered at -49 bp abolished promoter activity, establishing the importance of this sequence element. Single-base-pair changes in a C-box-like sequence centered at -77 bp also abolished promoter activity, but the pattern of mutational effects was different from that for the C box centered at -49 bp. Additional single-base-pair changes indicated that all 10 bp from -79 to -70 bp are important for Omega4403 promoter activity. Mutations at -59, -61, -62, and -63 bp also abolished promoter activity, defining a 5-bp element from -63 to -59 bp. This 5-bp element is separated from the 10-bp element (i.e., -79 to -70 bp) by 6 bp that can be changed without loss of promoter activity. Likewise, the 5 bp between the 5-bp element and the C box can be changed without loss of activity, but deletion of these 5 bp abolished activity, indicating that spacing is important. Sequences similar to the 5- and 10-bp elements, as well as the C box, are present in other C-signal-dependent promoters, suggesting some similarity in the regulatory mechanisms, but there are also indications that these cis elements do not function identically in the different promoters.

Bacterial Proteins↗

The IntP C-terminal segment is not required for excision of bacteriophage Mx8 from the Myxococcus xanthus chromosome.

During lysogenization of myxophage Mx8, phage DNA can be integrated into the attB site of the Myxococcus xanthus chromosome through site-specific recombination. We previously demonstrated that the Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the 112-amino-acid C-terminal segment of the IntP protein into a 13-amino-acid C-terminal segment of a new protein, IntR. To examine whether IntR is active for Mx8 excision, we have constructed a series of plasmids carrying various lengths of the intP-attP or intR-attR regions as well as the lacZ gene. The integrated Mx8 was excised at a high frequency, indicating that IntR is active for the excision. For Mx8 excision, a gene designated xis was shown to be required in addition to intR.

Bacteriophages↗

nsd, a locus that affects the Myxococcus xanthus cellular response to nutrient concentration.

Expression of the previously reported Tn5lac Omega4469 insertion in Myxococcus xanthus cells is regulated by the starvation response. Interested in learning more about the starvation response, we cloned and sequenced the region containing the insertion. Our analysis shows that the gene fusion is located in an open reading frame that we have designated nsd (nutrient sensing/utilizing defective) and that its expression is driven by a sigma70-like promoter. Sequence analysis of the nsd gene product provides no information on the potential structure or function of the encoded protein. In a further effort to learn about the role of nsd in the starvation response, we closely examined the phenotype of cells carrying the nsd::Tn5lac Omega4469 mutation. Our analysis showed that these cells initiate development on medium that contains nutrients sufficient to sustain vegetative growth of wild-type cells. Furthermore, in liquid media these same nutrient concentrations elicit a severe impairment of growth of nsd cells. The data suggest that the nsd cells launch a starvation response when there are enough nutrients to prevent one. In support of this hypothesis, we found that, when grown in these nutrient concentrations, nsd cells accumulate guanosine tetraphosphate, the cellular starvation signal. Therefore, we propose that nsd is used by cells to respond to available nutrient levels.

Base Sequence↗