Smaldone G.T., Jin Y., Whitfield D.L., Mu A.Y., Wong E.C., Wuertz S., Singer M. 2014. Growth of Myxococcus xanthus in continuous-flow-cell bioreactors as a method for studying development. Appl Environ Microbiol. 80(8):2461-7. [
Nutrient sensors and developmental timers are two classes of genes vital to the establishment of early development in the social soil bacterium Myxococcus xanthus. The products of these genes trigger and regulate the earliest events that drive the colony from a vegetative state to aggregates, which ultimately leads to the formation of fruiting bodies and the cellular differentiation of the individual cells. In order to more accurately identify the genes and pathways involved in the initiation of this multicellular developmental program in M. xanthus, we adapted a method of growing vegetative populations within a constant controllable environment by using flow cell bioreactors, or flow cells. By establishing an M. xanthus community within a flow cell, we are able to test developmental responses to changes in the environment with fewer concerns for effects due to nutrient depletion or bacterial waste production. This approach allows for greater sensitivity in investigating communal environmental responses, such as nutrient sensing. To demonstrate the versatility of our growth environment, we carried out time-lapse confocal laser scanning microscopy to visualize M. xanthus biofilm growth and fruiting body development, as well as fluorescence staining of exopolysaccharides deposited by biofilms. We also employed the flow cells in a nutrient titration to determine the minimum concentration required to sustain vegetative growth. Our data show that by using a flow cell, M. xanthus can be held in a vegetative growth state at low nutrient concentrations for long periods, and then, by slightly decreasing the nutrient concentration, cells can be allowed to initiate the developmental program.
Bragg J., Rajkovic A., Anderson C., Curtis R., Van Houten J., Begres B., Naples C., Snider M., Fraga D., Singer M. 2012. Identification and characterization of a putative arginine kinase homolog from Myxococcus xanthus required for fruiting body formation and cell differentiation. J Bacteriol. 194(10):2668-76. [
Arginine kinases catalyze the reversible transfer of a high-energy phosphoryl group from ATP to l-arginine to form phosphoarginine, which is used as an energy buffer in insects, crustaceans, and some unicellular organisms. It plays an analogous role to that of phosphocreatine in vertebrates. Recently, putative arginine kinases were identified in several bacterial species, including the social Gram-negative soil bacterium Myxococcus xanthus. It is still unclear what role these proteins play in bacteria and whether they have evolved to acquire novel functions in the species in which they are found. In this study, we biochemically purified and characterized a putative M. xanthus arginine kinase, Ark, and demonstrated that it has retained the ability to catalyze the phosphorylation of arginine by using ATP. We also constructed a null mutation in the ark gene and demonstrated its role in both certain stress responses and development.
Rosario C.J., Singer M. 2010. Developmental expression of dnaA is required for sporulation and timing of fruiting body formation in Myxococcus xanthus. Mol. Microbiol. 76(5):1322-33. [
Inhibition of DNA replication within the first 6 h of development results in a block in the developmental programme in the social soil bacterium Myxococcus xanthus. We have interpreted these data to imply that M. xanthus requires a new round of DNA replication early in the developmental programme. To further understand the role of DNA replication during development in M. xanthus we focused on the regulation of dnaA which encodes the initiator protein of DNA replication. In this work, we demonstrate that immediately upon nutrient deprivation dnaA (MXAN1001) transcript levels decrease to 10-15% of vegetative levels and then transiently increase between 4 and 6 h post initiation. This expression is dependent on several early developmental regulators, including relA (MXAN3204), sigD (MXAN2957) and sdeK (MXAN1014). It is also dependent upon an 85 bp region located just upstream to the dnaA promoter. Our data suggest that while developmental dnaA expression is not essential for development, its expression allows for the proper timing and maximum efficiency of the sporulation process. In addition, we speculate that developmental control of dnaA expression may provide a mechanism for predetermination of cell fate during the differentiation process.
Wu D, Hugenholtz P, Mavromatis K, Pukall R, Dalin E, Ivanova NN, Kunin V, Goodwin L, Wu M, Tindall BJ, Hooper SD, Pati A, Lykidis A, Spring S, Anderson IJ, D'haeseleer P, Zemla A, Singer M, Lapidus A, Nolan M, Copeland A, Han C, Chen F, Cheng JF, Lucas S, Kerfeld C, Lang E, Gronow S, Chain P, Bruce D, Rubin EM, Kyrpides NC, Klenk HP, Eisen JA. 2009. A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea. Nature. 462(7276):1056-60. [
Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic 'phylogenomic' efforts to compile a phylogeny-driven 'Genomic Encyclopedia of Bacteria and Archaea' in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.
Bode H.B., Ring M.W., Schwär G., Altmeyer
M.O., Kegler C., Jose I.R., Singer M., Müller R. 2009. Identification
of Additional Players in the Alternative Biosynthesis Pathway to Isovaleryl-CoA
in the Myxobacterium Myxococcus xanthus. Chembiochem. 10(1):128-40. [
Isovaleryl-CoA (IV-CoA) is usually derived from the degradation of leucine by using the Bkd (branched-chain keto acid dehydrogenase) complex. We have previously identified an alternative pathway for IV-CoA formation in myxobacteria that branches from the well-known mevalonate-dependent isoprenoid biosynthesis pathway. We identified 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase (MvaS) to be involved in this pathway in Myxococcus xanthus, which is induced in mutants with impaired leucine degradation (e.g., bkd(-)) or during myxobacterial fruiting-body formation. Here, we show that the proteins required for leucine degradation are also involved in the alternative IV-CoA biosynthesis pathway through the efficient catalysis of the reverse reactions. Moreover, we conducted a global gene-expression experiment and compared vegetative wild-type cells with bkd mutants, and identified a five-gene operon that is highly up-regulated in bkd mutants and contains mvaS and other genes that are directly involved in the alternative pathway. Based on our experiments, we assigned roles to the genes required for the formation of IV-CoA from HMG-CoA. Additionally, several genes involved in outer-membrane biosynthesis and a plethora of genes encoding regulatory proteins were decreased in expression levels in the bkd(-) mutant; this explains the complex phenotype of bkd mutants including a lack of adhesion in developmental submerse culture.
Rosario C.J., Singer M. 2007. The Myxococcus xanthus
developmental program can be delayed by inhibition of DNA replication. J. Bacteriol. 189:8793-8800. [
Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB(A116V), was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37 degrees C. We demonstrated that at the nonpermissive temperature the DnaB(A116V) mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB(A116V) mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.
Ossa F.*, Diodati M.E.*, Caberoy N.B., Giglio K.M., Edmonds M.,
Singer M., Garza A.G. 2007. The Myxococcus xanthus Nla4
protein is important for expression of stringent response-associated genes,
ppGpp accumulation, and fruiting body development. J. Bacteriol.
Changes in gene expression are important for the landmark morphological events that occur during Myxococcus xanthus fruiting body development. Enhancer binding proteins (EBPs), which are transcriptional activators, play prominent roles in the coordinated expression of developmental genes. A mutation in the EBP gene nla4 affects the timing of fruiting body formation, the morphology of mature fruiting bodies, and the efficiency of sporulation. In this study, we showed that the nla4 mutant accumulates relatively low levels of the stringent nucleotide ppGpp. We also found that the nla4 mutant is defective for early developmental events and for vegetative growth, phenotypes that are consistent with a deficiency in ppGpp accumulation. Further studies revealed that nla4 cells produce relatively low levels of GTP, a precursor of RelA-dependent synthesis of (p)ppGpp. In addition, the normal expression patterns of all stringent response-associated genes tested, including the M. xanthus ppGpp synthetase gene relA, are altered in nla4 mutant cells. These findings indicate that Nla4 is part of regulatory pathway that is important for mounting a stringent response and for initiating fruiting body development.
Diodati M.E., Gill R.E., Plamann L., Singer M. 2007.
"Initiation and Early Developmental Events." Myxobacteria: Multicellularity
and Differentiation (Peer-Reviewed Book Chapter) 43-76.
Suen, G., J.S. Jakobsen, B.S. Goldman, M. Singer, A.G. Garza and R.D.
Welch 2006. Bacterial Postgenomics: the Promise and Peril of Systems Biology
J. Bacteriol. 188:7999-8004 [
Pham V.D., C.W. Shebelut, I.R. Jose, D. A. Hodgson, D.E. Whitworth and M. Singer. The response regulator PhoP4 is required for late developmental events in Myxococcus xanthus. 2006. Microbiology. 152:1609-20 [ pdf ]
Phosphate regulation is complex in the developmental prokaryote Myxococcus xanthus, and requires at least four two-component systems (TCSs). Here, the identification and characterization of a member of one TCS, designated PhoP4, is reported. phoP4 insertion and in-frame deletion strains caused spore viability to be decreased by nearly two orders of magnitude, and reduced all three development-specific phosphatase activities by 80-90 % under phosphate-limiting conditions. Microarray and quantitative PCR analyses demonstrated that PhoP4 is also required for appropriate expression of the predicted pstSCAB-phoU operon of inorganic phosphate assimilation genes. Unlike the case for the other three M. xanthus Pho TCSs, the chromosomal region around phoP4 does not contain a partner histidine kinase gene. Yeast two-hybrid analyses reveal that PhoP4 interacts reciprocally with PhoR2, the histidine kinase of the Pho2 TCS; however, the existence of certain phenotypic differences between phoP4 and phoR2 mutants suggests that PhoP4 interacts with another, as-yet unidentified, histidine kinase.
Viswanathan, P., M. Singer and L. Kroos. 2006. Role of σD in Regulating Genes and Signals during Myxococcus xanthus Development. J. Bacteriol. 188:3246-3256 [ pdf ]
Starvation-induced development of Myxococcus xanthus is an excellent model for biofilm formation because it involves cell-cell signaling to coordinate formation of multicellular mounds, gene expression, and cellular differentiation into spores. The role of σD, an alternative σ factor important for viability in stationary phase and for stress responses, was investigated during development by measuring signal production, gene expression, and sporulation of a sigD null mutant alone and upon codevelopment with wild-type cells or signaling mutants. The sigD mutant responded to starvation by inducing (p)ppGpp synthesis normally but was impaired for production of A-signal, an early cell density signal, and for production of the morphogenetic C-signal. Induction of early developmental genes was greatly reduced, and expression of those that depend on A-signal was not restored by codevelopment with wild-type cells, indicating that σD is needed for cellular responses to A-signal. Despite these early developmental defects, the sigD mutant responded to C-signal supplied by codeveloping wild-type cells by inducing a subset of late developmental genes. σD RNA polymerase is dispensable for transcription of this subset, but a distinct regulatory class, which includes genes essential for sporulation, requires σD RNA polymerase or a gene under its control, cell autonomously. The level of sigD transcript in a relA mutant during growth is much lower than in wild-type cells, suggesting that (p)ppGpp positively regulates sigD transcription in growing cells. The sigD transcript level drops in wild-type cells after 20 min of starvation and remains low after 40 min but rises in a relA mutant after 40 min, suggesting that (p)ppGpp negatively regulates sigD transcription early in development. We conclude that σD synthesized during growth occupies a position near the top of a regulatory hierarchy governing M. xanthus development, analogous to σ factors that control biofilm formation of other bacteria.
Tzeng, L., T.N. Ellis and M. Singer. 2006. DNA Replication during Aggregation Phase Is Essential for Myxococcus xanthus Development. J. Bacteriol. 188:2774-79 [ pdf ]
Previous studies have demonstrated that fruiting body-derived Myxococcus xanthus myxospores contain two fully replicated copies of its genome, implying developmental control of chromosome replication and septation. In this study, we employ DNA replication inhibitors to determine if chromosome replication is essential to development and the exact time frame in which chromosome replication occurs within the developmental cycle. Our results show that DNA replication during the aggregation phase is essential for developmental progression, implying the existence of a checkpoint that monitors chromosome integrity at the end of the aggregation phase.
Diodati, M.E., F. Ossa, N.B. Caberoy, I.R. Jose, W. Hiraiwa, M.M. Igo, M. Singer and A.G. Garza. 2006. Nla18, a Key Regulatory Protein Required for Normal Growth and Development of Myxococcus xanthus. J. Bacteriol. 188:1733-43 [ pdf ]
NtrC-like activators regulate the transcription of a wide variety of adaptive genes in bacteria. Previously, we demonstrated that a mutation in the ntrC-like activator gene nla18 causes defects in fruiting body development in Myxococcus xanthus. In this report, we describe the effect that nla18 inactivation has on gene expression patterns during development and vegetative growth. Gene expression in nla18 mutant cells is altered in the early stages of fruiting body development. Furthermore, nla18 mutant cells are defective for two of the earliest events in development, production of the intracellular starvation signal ppGpp and production of A-signal. Taken together, these results indicate that the developmental program in nla18 mutant cells goes awry very early. Inactivation of nla18 also causes a dramatic decrease in the vegetative growth rate of M. xanthus cells. DNA microarray analysis revealed that the vegetative expression patterns of more than 700 genes are altered in nla18 mutant cells. Genes coding for putative membrane and membrane-associated proteins are among the largest classes of genes whose expression is altered by nla18 inactivation. This result is supported by our findings that the profiles of membrane proteins isolated from vegetative nla18 mutant and wild-type cells are noticeably different. In addition to genes that code for putative membrane proteins, nla18 inactivation affects the expression of many genes that are likely to be important for protein synthesis and gene regulation. Our data are consistent with a model in which Nla18 controls vegetative growth and development by activating the expression of genes involved in gene regulation, translation, and membrane structure.
Tzeng L. and M. Singer. 2005. DNA replication during sporulation in Myxococcus xanthus fruiting bodies. PNAS 102:14428-33 [ pdf ]
During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.
Pham V.D., C.W. Shebelut, B. Mukherjee and M. Singer. 2005. RasA is required for Myxococcus xanthus development and social motility. J. Bacteriol. 187:6845-48 [ pdf ]
An insertion in the rasA gene entirely blocked developmental aggregation and sporulation in Myxococcus xanthus while also reducing swarm expansion on a 0.3% agar surface. Data presented here demonstrate that rasA is required for extracellular fibril formation and social gliding motility.
Pham V.D., C.W. Shebelut, E.J. Zumstein and M. Singer. 2005. BrgE is a regulator of Myxococcus xanthus development. Molecular Microbiology. 57:762-73. [ pdf ], [ erratum ]
We report here the identification and characterization of a member of the Myxococcus xanthus SdeK signal transduction pathway, BrgE. This protein was identified as an SdeK-interacting component using a yeast two-hybrid screen, and we further confirmed this interaction by the glutathione S-transferase (GST) pulldown assay. Additional yeast two-hybrid analyses revealed that BrgE preferentially interacts with the putative amino-terminal sensor domain of SdeK, but not with the carboxy-terminal kinase domain. A brgE insertion strain was shown to be blocked in development between aggregation and mound formation, and decreased by 50-fold in viable spore production compared with the parental wild type. These phenotypes are similar to those of sdeK mutants. The brgE mutation also altered expression of a sample of Tn5 lac developmental markers that are also SdeK regulated. Finally, we demonstrated that a brgE sdeK double mutant has a more severe sporulation defect than either of the two single mutants, suggesting that BrgE and SdeK act synergistically to regulate wild-type levels of sporulation. In sum, these data suggest that BrgE operates as an auxiliary factor to stimulate the SdeK signal transduction pathway by directly binding to the amino-terminal sensor domain of SdeK.
Pham V.D., C.W. Shebelut, M.E. Diodati, C.T. Bull and M. Singer. 2005. Mutations affecting predation ability of the soil bacterium Myxococcus xanthus. Microbiology. 151:1865-74. [ pdf ]
Myxococcus xanthus genetic mutants with characterized phenotypes were analysed for the ability to prey on susceptible bacteria. Quantification of predatory ability was scored by a newly developed method under conditions in which prey bacteria provided the only source of nutrients. These results were corroborated by data derived using a previously published protocol that measures predation in the presence of limited external nutrients. First, early developmental regulatory mutants were examined, because their likely functions in assessing the local nutrient status were predicted to be also important for predation. The results showed that predation efficiency is reduced by 64-80 % for mutants of three A-signalling components, AsgA, AsgC and AsgE, but not for AsgB. This suggests that an Asg regulon function that is separate from A-signal production is needed for predation. Besides the Asg components, mutations in the early developmental genes sdeK and csgA were also consistently observed to reduce predatory efficacy by 36 and 33 %, respectively. In contrast, later developmental components, such as DevRS, 4406 and PhoP4, did not appear to play significant roles in predation. The predatory abilities of mutants defective for motility were also tested. The data showed that adventurous, but not social, motility is required for predation in the assay. Also, mutants for components in the chemotaxis-like Frz system were found to be reduced in predation efficiency by between 62 and 85 %. In sum, it was demonstrated here that defects in development and development-related processes affect the ability of M. xanthus to prey on other bacteria.
Brenner, M., A. Garza and M. Singer. 2004. nsd,
a Locus that Affects the Myxococcus xanthus Cellular Response to Nutrient Concentration. J. Bacteriol. 186:3461-3471. [ pdf ]
Expression of the previously reported Tn5lacΩ4469 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 σ70-like promoter. Sequence analysis of the nsd gene
product provides no information on 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::Tn5lacΩ4469 mutation. Our analysis
showed that these cells initiate development on media 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 from nsd cells. The data suggests 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 ppGpp, the cellular starvation signal.
Therefore, we propose that nsd is used by cells to respond to available
Pollack, J. and M. Singer. 2001. SdeK, a Histidine Kinase Required
for Myxococcus xanthus Development. J. Bacteriol.. 183:3589-3596. [ pdf ]
The sdeK gene is essential to the Myxococcus xanthus
developmental process. We reported previously, based on sequence analysis
(A.G. Garza, J. S. Pollack, B. Z. Harris, A. Lee, I. M. Keseler, E.F.
Licking, and M. Singer. J. Bacteriol. 180:4628:4637, 1998), that
SdeK appears to be a histidine kinase. In the present study, we have conducted
both biochemical and genetic analyses to test the hypothesis that SdeK
is a histidine kinase. An SdeK fusion protein containing an N-terminal
polyhistidine tag (His-SdeK) displays the biochemical characteristics
of a histidine kinase. Furthermore, a histidine 286 of SdeK, the putative
site of phosphorylation, is required for both in vitro and in vivo protein
activity. The results of these assays have led us to conclude that SdeK
is indeed a histidine kinase. The developmental phenotype of a ΔsdeK1
strain could not be rescued by codevelopment with wild-type cells, indicating
that the defect is not due to the mutant's inability to produce an extracellular
signal. Furthermore, the ΔsdeK1 mutant was found to produce
both A- and C-signal, based on A-factor and codevelopment assays with
csgA mutant, respectively. The expression patterns of several
Tn5lacz transcriptional fusions were examined in the ΔsdeK1-null
background, and we found that all C-signal-dependent fusions assayed also
required SdeK for full expression. Our results indicate that SdeK is a
histidine kinase that is part of a signal transduction pathway which,
in concert with the C-signal transduction pathway, controls the activation
of developmental-gene expression required to progress past the aggregation
Garza, A.G., B.Z. Harris, J.S. Pollack and M. Singer. 2000. The asgE
locus is required for cell-cell signaling during the Myxococcus xanthus
development. Mol. Microbiol. 35:812-824. [ pdf ]
In response to starvation, Myxococcus xanthus undergoes a multicellular
developmental process that produces a dome-shaped fruiting body structure filled
with differentiated cells called myxospores. Two insertion mutants that block
the final stages of fruiting body morphogenesis and reduce sporulation efficiency
were isolated and characterized. DNA sequence analysis revealed that the chromosomal
insertions are located in open reading frames ORFG2 and asgE, which
are separated by 68 bp. The sporulation defect of cells carrying the asgE
insertion can be rescued phenotypically when co-developed with wild-type cells,
whereas the sporulation efficiency of cells carrying the ORF2 insertion was
not improved when mixed with wild-type cells. Thus, the asgE insertion
mutant appears to belong to a class of developmental mutants that are unable
to produce cell-cell signals required for M. xanthus development, but
they retain the ability to respond to them when they are provided by wild-type
cells. Several lines of evidence indicate that asgE cells fail to produce
normal levels of A-factor, a cell density signal. A-factor consists of a mixture
of heat-stable extracellular proteases. The asgE mutant yielded about
10-fold less heat labile A-factor and about twofold less heat-stable A-factor
than wild-type cells, suggesting that the primary defect of asgE cells
is in the production or release of heat-labile A-factor.
Garza A.G., B.Z. Harris, B. Greenberg, and M. Singer. 2000. Control
of asgE Expression during Growth and Development of Myxococcus xanthus.
J. Bacteriol. 182:6622-6629. [ pdf ]
One of the earliest events in the Myxococcus xanthus developmental cycle
is production of an extracellular cell density called A-signal (or A-factor).
Previously, we showed that cells carrying an insertion in the asgE gene
fail to produce normal levels of this cell-cell signal. In this study we found
that expression of asgE is growth phase regulated and developmentally
regulated. Several lines of evidence indicate that asgE is cotranscribed
with an upstream gene during development. Using primer extension analyses, we
identified two 5' ends for this developmental transcript. The DNA sequence upstream
of one 5' end has similarity to the promoter regions of server al genes that
are A-signal dependent, whereas sequences located upstream of the second 5'
end show similarity to promoter elements identified for genes that are C-signal
dependent. Consistent with this result is our finding that mutants failing to
produce A-signal or C-signal are defective for developmental expression of asgE.
In contrast to developing cells, the large majority of the asgE transcript
found in vegetative cells appears to be monocistronic. This finding suggest
that asgE uses different promoters for expression indicating that this
vegetative promoter is induced by starvation. The data presented here, in combination
with our previous results, indicate that the level of AsgE in vegetative cells
is sufficient for this protein to carry out its function during development.
Garza A.G., J.S. Pollack, B.Z. Harris, A. Lee, I.M. Keseler, E.F.
Licking, and M. Singer. 1998. SdeK Is Required for Early Fruiting
Body Development in Myxococcus xanthus. J. Bacteriol.
180:4628-4637. [ pdf ]
Myxococcus xanthus cells carrying the Ω4408 Tn5lac
insertion at the sde locus show defects in fruiting body development
and sporulation. Our analysis of sde expression patterns showed
that this locus is induced early in the development program (0 to 2 h)
and that expression increases approximately fivefold after 12 h of development.
Further studies showed that expression of sde is induced as growing
cells enter stationary phase, suggesting that activation of the sde
locus is not limited to the developmental process. Because the peak levels
of sde expression in both an sde+ and an sde mutant
background were similar, we conclude that the sde locus is not
autoregulated. Characterization of the sde locus by DNA sequence
analysis localized the 5' end of sde transcript to a guanine nucleotide
307 bp upstream of the proposed start for the SdeK coding sequence. The
DNA sequence in the -12 and -24 regions upstream of the sde transcriptional
start site shows similarity to the σ54 family of promoters. The results
of complementation studies suggest that the defects in development and
sporulation was found to have similarity to the sequences of the histidine
protein kinases of two-component regulatory systems. Based on our results,
we propose that SdeK may be part of a signal transduction pathway required
for the activation and propagation of the early developmental program.
Harris, B., Kaiser D., and M. Singer. 1998. The guanosine nucleotide
(p)ppGpp initiates development and A-factor production in Myxococcus xanthus.
Genes & Dev. 12:1022-1035. [ pdf ]
Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in
response to amino acid limitation, induce early gene expression leading to multicellular
fruiting body formation in Myxococcus xanthus. A mutant (DK527) that
fails to accumulate (p)ppGpp in response to starvation was found to be blocked
in development prior to aggregation. By use of a series of developmentally regulated
Tn5lac transcriptional fusion reporters, the time of developmental
arrest in DK527 was narrowed to within the few hours of development, the period
of starvation recognition. The mutant is also defective in the production of
A-factor, an early extracellular cell-density signal. The relA gene
from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp
synthetase, rescues this mutant. We also demonstrate that inactivation of the
M. xanthus relA homolog blocks development and the accumulation of
(p)ppGpp. Moreover, the wild-type allele of myxococcus relA resnocues
DK527. These observations support a model in which accumulation of (p)ppGpp,
in response to starvation, initiates the program of fruiting body development,
including the production of A-factor.
Harris, B., and M. Singer. 1998. Identification and Characterization
of the Myxococcus xanthus argE Gene. J. Bacteriol. 180:6412-6414. [ pdf ]
The chromosomal acetylornithine deacetylase (argE) gene of Myxococcus
xanthus was identified via homology to acetylornithine deacetylases from
other bacterial species. A mutant carrying a disruption in argE was
unable to grow on minimal media lacking supplemental arginine and formed fruiting
bodies and spores in response to arginine starvation at high cell density.
Singer, M., and D. Kaiser. 1995. Ectopic production of guanosine penta-
and tetraphosphate can initiate early developmental gene expression in Myxococcus
xanthus. Genes & Dev. 9:1633-1644.
Amino acid or carbon limitation is sufficient to initiate fruiting body development
in Myxococcus xanthus. In both Escherichia coli and M. xanthus the levels
of guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp] rise transiently
when cells are starved for amino acids or carbon. Ectopic increase in the intracellular
concentration of (p)ppGpp was achieved in M. xanthus by introducing a copy of
the E. coli relA gene, whose product catalyzes pyrophosphate transfer from ATP-
to GTP- forming pppGpp. The E. coli RelA protein was detected in these M. xanthus
strains, and a rise in (p)ppGpp was observed chromatographically. This increase
in the intracellular (p)ppGpp levels was sufficient to activate developmentally
specific gene expression. Although (p)ppGpp is made from GTP, the intracellular
GTP pool from these strains was not significantly decreased. Moreover, when
the GTP pool was lowered by either of two specific inhibitors of GTP synthesis,
mycophenolic acid or decoyinine, development was not induced. These results
suggest that M. xanthus cells can assess their nutritional status by
monitoring the internal availability of amino acids through (p)ppGpp levels.
Stephens, C., M. Singer and L. Shapiro. 1994. An ATP/ADP switch.
Current Biology. 4:630-632.
Regulatory factors that initiate forespore-specific transcription during Bacillus
subtilis sporulation respond to adenosine nucleotide ratios.
Singer, M., D.J. Jin, W.A. Walter and C.A. Gross. 1993. Genetic Evidence
for the Interaction between Cluster I and Cluster III Rifampicin Resistant Mutations.
J. Mol Biol. 231:1-5. [ pdf ]
Rifampicin-resistant (Rifr) mutations of Escherichia coli
map to the central portion of the rpoB gene, which encodes the ß
subunit of RNA polymerase. These mutations are located in three distinct clusters,
designated I, II and III. Three intragenic suppressors of the cluster III Rifr
mutation, rpoB3406(RH687), restore the ability of the mutant strain
to grow at low and high temperatures and map to a single locus in cluster I.
These suppressors are identical to two previously characterized Rifr
alleles, rpoB3401(RC529) and rpoB3402(RS529). None of the
other 14 previously identified Rifr mutations that we have characterized
confers this phenotype. We suggest that this allele-specific suppression results
from interaction between Cluster I and Cluster III of that ß subunit.
Singer, M., W.A. Walter, B.M. Cali, P. Rouviere, H.H. Liebke, R.L. Gourse
and C.A. Gross. 1991. Physiological Effects of the Fructose-1,6-Diphosphate
Aldolase ts8 Mutation on Stable RNA Synthesis in Escherichia coli.
J. Bacteriol. 173:6249-6257.
The conditional lethal mutations ts8 and h8 are located in
fda, the gene encoding aldolase, and they inhibit RNA synthesis upon
shift to the nonpermissive temperature. We demonstrate that both mutations preferentially
inhibit stable RNA synthesis and that this inhibition occurs at the level of
transcription initiation. The susceptibility of a promoter to the inhibitory
effects of ts8 is correlated with the ability of the promoter to be
growth rate regulated. This effect is independent of relA and spoT
function. Inhibition is dependent upon glucose metabolism past the generation
of glucose-6-phosphate; however, the mechanism of this effects is unknown.
Singer, M., P. Rossmiessl, B.M. Cali, H. Liebke and C.A. Gross. 1991.
The Escherichia coli ts8 Mutation is an Allele of fda, the
Gene Encoding Fructose-1,6-Diphosphate Aldolase. J. Bacteriol. 173:6242-6248.
The ts8 mutant of Escherichia coli has previously been shown
to preferentially inhibit stable RNA synthesis when shifted to the nonpermissive
temperature. We demonstrate in this report that the ts8 mutation is
an allele of fda, the gene that encodes the glycolytic enzyme frutose-1,6-diphosphate
aldolase. We show that ts8 and a second fda mutation, h8,
isolated and characterized by A. Bock and F.C. Neidhardt, are dominant mutations
and that they encode a thermolabile aldolase activity.