Identification and characterization of genes required for early Myxococcus xanthus developmental gene expression

Starvation and cell density regulate the developmental expression of Myxococcus xanthus gene 4521. Three classes of mutants allow expression of this developmental gene during growth on nutrient agar, such that colonies of strains containing a Tn5 lac Omega4521 fusion are Lac(+). One class of these mutants inactivates SasN, a negative regulator of 4521 expression; another class activates SasS, a sensor kinase-positive regulator of 4521 expression; and a third class blocks lipopolysaccharide (LPS) O-antigen biosynthesis. To identify additional positive regulators of 4521 expression, 11 Lac(-) TnV.AS transposon insertion mutants were isolated from a screen of 18,000 Lac(+) LPS O-antigen mutants containing Tn5 lac Omega4521 (Tc(r)). Ten mutations identified genes that could encode positive regulators of 4521 developmental expression based on their ability to abolish 4521 expression during development in the absence of LPS O antigen and in an otherwise wild-type background. Eight of these mutations mapped to the sasB locus, which encodes the known 4521 regulators SasS and SasN. One mapped to sasS, whereas seven identified new genes. Three mutations mapped to a gene encoding an NtrC-like response regulator homologue, designated sasR, and four others mapped to a gene designated sasP. One mutation, designated ssp10, specifically suppressed the LPS O-antigen defect; the ssp10 mutation had no effect on 4521 expression in an otherwise wild-type background but reduced 4521 developmental expression in the absence of LPS O antigen to a level close to that of the parent strain. All of the mutations except those in sasP conferred defects during growth and development. These data indicate that a number of elements are required for 4521 developmental expression and that most of these are necessary for normal growth and fruiting body development.     

Inhibition of Development of Myxococcus xanthus by Eukaryotic Protein Kinase Inhibitors

Myxococcus xanthus is a social bacterium that lives in the soil and undergoes spectacular development to form multicellular fruiting bodies. It contains a large family of eukaryote-like serine/threonine protein kinases. We found that a number of inhibitors for eukaryotic protein serine, threonine, and tyrosine kinases could inhibit the development and sporulation of M. xanthus to various degrees. These results suggest that serine/threonine and tyrosine phosphorylation may be involved in development of M. xanthus. None of the inhibitors tested had any effect on vegetative growth of M. xanthus. Most of them seemed to act during the early stages of development. However, the expression of a very early development-specific gene, Ω4521, was not significantly affected by the inhibitors. The patterns of protein phosphorylation during development were also not significantly altered by the inhibitors, suggesting that the targets of the inhibitors are minor or unstable phosphoproteins but play key roles in fruiting-body formation in M. xanthus.     

Methylation of FrzCD Defines a Discrete Step in the Developmental Program of Myxococcus xanthus

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

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°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.     

Synergism between morphogenetic mutants of Myxococcus xanthus.

Myxococcus xanthus, a social procaryotic microorganism, forms fruiting bodies and myxospores. We have isolated a collection of mutants of M. xanthus that are defective in fruiting morphogenesis and have studied synergistic interaction in pairwise mixtures of these mutants. Certain pairs of these fruiting-defective mutants can fruit when mixed together. Similarly, certain mutants that cannot sporulate under standard fruiting conditions can form myxospores in the presence of wildtype or other nonsporulating mutants. The pattern of synergism between pairs of conditional nonsporulating mutants defines at least three and probably four groups of mutants, such that members of a group cannot synergize with each other but can synergize with members of other groups.     

Genes required for both gliding motility and development in Myxococcus xanthus

Myxococous xanthus cells can glide both as individual cells, dependent on A dventurous motility (A motility), and as groups of cells, dependent upon S ocial motility (S motility), Tn5-lac mutagenesis was used to generate 16 new A^- and nine new S^- mutations. In contrast with previous results, we find that subsets of A^- mutants are defective in fruiting body morphogenesis and/or myxospore differentiation. All S^- mutants are defective in fruiting body morphogenesis, consistent with previous results. Whereas some S^- mutants produce a wild-type complement of spores, others are defective in the differentiation of myxospores. Therefore, a subset of the A genes and all of the S genes are critical for fruiting body morphogenesis. Subsets of both A and S genes are essential for sporulation. Three S::Tn5–lac insertions result in surprising phenotypes. Colonies of two S^- mutants glide on ‘swim’ (0.35% agar) plates to form fractal patterns. These S^- mutants are the first examples of a bacterium in which mutations result in fractal patterns of colonial spreading. An otherwise wild-type strain with one S^- insertion resembles the frz^- sglA1^- mutants upon development, suggesting that this S^- gene defines a new chemotaxis component in M. xanthus.     

Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Antibiotic Production by Erwinia herbicola Eh1087: Its Role in Inhibition of Erwinia amylovora and Partial Characterization of Antibiotic Biosynthesis Genes

Mutants of Erwinia herbicola Eh1087 (Ant⁻), which did not produce antibiotic activity against Erwinia amylovora, the fire blight pathogen, were selected after TnphoA mutagenesis. In immature pear fruit Ant⁻ mutants grew at the same rate as wild-type strain Eh1087 but did not suppress development of the disease caused by E. amylovora. These results indicated that antibiosis plays an important role in the suppression of disease by strain Eh1087. All of the Ant⁻ mutations obtained were located in a 2.2-kb region on a 200-kb indigenous plasmid. Sequence analysis of the mutated DNA region resulted in identification of six open reading frames, designated ORF1 through ORF6, four of which were essential to antibiotic expression. One gene was identified as a gene which encodes a translocase protein which is probably involved in antibiotic secretion. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of plasmid proteins produced in Escherichia coli minicells confirmed the presence of proteins whose sizes corresponded to the sizes of the predicted open reading frame products.     

Suppressors that permit A-signal-independent developmental gene expression in Myxococcus xanthus.

Progression through the early stages of Myxococcus xanthus fruiting body development requires the cell-to-cell transmission of soluble material called A signal. During these early stages, expression from the gene identified by Tn5 lac insertion omega 4521 increases. A DNA probe of the omega 4521 gene was constructed. Use of this probe showed that accumulation of mRNA corresponding to the omega 4521 gene depends upon A signal. A-signal-deficient (asg) mutants fail to accumulate this RNA, and the external addition of A signal restores accumulation. To identify links between A signal and its responsive gene, omega 4521, suppressors of an asg mutation were generated. All of the suppressor alleles restored lacZ expression from omega 4521 in the absence of A signal, and they were demonstrated to be neither reversions of the asgB mutation nor mutations in the promoter of omega 4521. Fifteen suppressor mutations map to two loci, sasA and sasB (for suppressor of asg). sasA and sasB mutants differ phenotypically during growth and development. Mid-logarithmic-phase sasA asgB double mutants, like sas+ asg+ strains, express low levels of lacZ, whereas sasB asgB double mutants express high levels. sasA asg+ mutants form abnormal colonies, are less cohesive than wild type, and are defective in fruiting body formation and sporulation. In contrast, sasB asg+ mutants form normal colonies, are as cohesive as wild type, and appear to develop normally. The characteristics of sasA suppressors implicate the sasA+ product as a negative regulator in the A-signal-dependent regulation of omega 4521.     

Targeted disruption of fad24, a regulator of adipogenesis,causes pre-implantation embryonic lethality due to the growth defect at the blastocyst stage

In previous studies, we identified a novel gene, factor for adipocyte differentiation 24 (fad24), which plays an important role during the early stages of adipogenesis in mouse 3T3-L1 cells. Moreover, overexpression of fad24 increased the number of smaller adipocytes in white adipose tissue and improved glucose metabolic activity in mice, thus indicating that fad24 functions as a regulator of adipogenesis in vivo. However, the physiological roles of fad24 in vivo are largely unknown. In this study, we attempted to generate fad24-deficient mice by gene targeting. No fad24-null mutants were recovered after embryonic day 9.5 (E9.5). Although fad24-null embryos were detected in an expected Mendelian ratio of genotypes at E3.5, none of the homozygous mutants developed into blastocysts. In vitro culture experiments revealed that fad24-null embryos develop normally to the morula stage but acquire growth defects during subsequent stages. The number of nuclei decreased in fad24-deficient morulae compared with that in wild-type ones. These results strongly suggested that fad24 is essential for pre-implantation in embryonic development, particularly for the progression to the blastocyst stage.     

The aadA Gene of Plasmid R100 Confers Resistance to Spectinomycin and Streptomycin in Myxococcus xanthus

Plasmids with the aadA gene from plasmid R100, which confers resistance to the aminoglycosides spectinomycin and streptomycin in Escherchia coli, can be introduced into wild-type Myxococcus xanthus, strain DK1622, by electroporation. Recombinant M. xanthus strains with integrated plasmids carrying the aadA gene acquire resistance to high levels of these antibiotics. Selection for aadA in M. xanthus can be carried out independently of, or simultaneously with, selection for resistance to kanamycin. The kinds and frequencies of recombination events observed between integrative plasmids with aadA and the M. xanthus chromosome are similar to those observed after the transformation of yeast. Cleavage of integrative plasmid DNA at a site adjacent to a region of homology between the plasmid and the M. xanthus genome favors the targeted disruption of M. xanthus genes by allele replacement.     

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Two gene systems control movement

Summary A large number of motility mutants of the gliding bacterium Myxococcus xanthus have been isolated and analyzed by transduction. Almost all nonmotile mutants are found to be double mutants. This is explained by the existence of two parallel and almost independent multi-gene systems controlling motility, in which case at least one mutation in each system would be required eliminate motility. Only one locus, called mgl, has been found to be shared by both systems. Wild type cells move singly and in groups. Single cells move if they carry a complete gene system A, the genes of which are described in the preceding paper. Groups of cells can move if they carry a complete gene system S, but single A^–S⁺ cells do not move. Linkage analysis reveals at least 9 different loci in system S. One class of S^– mutants, those mutated in the locus tgl, are conditional mutants which, after contact with tgl ⁺ cells, become temporarily motile as cell groups. Most system A mutations have little effect on fruiting but many system S mutations block it, suggesting that system S plays a role in the fruiting process.     

Growth of Myxococcus xanthus in Continuous-Flow-Cell Bioreactors as a Method for Studying Development

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.     

Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation enhances its intracellular growth within macrophages

Modification of lipid A is essential for bacterial adaptation to its host. Salmonella Typhimurium LpxR potentially detoxifies lipid A by 3′-O-deacylation; however, the involvement of deacylation in its adaptation remains unclear. LpxR-dependent 3′-O-deacylation was observed in the stationary phase. When macrophages were infected with stationary phase bacteria, the intracellular growth of the lpxR-null strain was lower than that of the wild-type strain. Furthermore, the expression level of inducible nitric oxide synthase was higher in the cells infected with the lpxR-null strain than in the cells infected with the wild-type strain. These results indicate that lipid A 3′-O-deacylation is beneficial for intracellular growth.     

Genetic interactions of smc, ftsK, and parB genes in Streptomyces coelicolor and their developmental genome segregation phenotypes

The mechanisms by which chromosomes condense and segregate during developmentally regulated cell division are of interest for Streptomyces coelicolor, a sporulating, filamentous bacterium with a large, linear genome. These processes coordinately occur as many septa synchronously form in syncytial aerial hyphae such that prespore compartments accurately receive chromosome copies. Our genetic approach analyzed mutants for ftsK, smc, and parB. DNA motor protein FtsK/SpoIIIE coordinates chromosome segregation with septum closure in rod-shaped bacteria. SMC (structural maintenance of chromosomes) participates in condensation and organization of the nucleoid. ParB/Spo0J partitions the origin of replication using a nucleoprotein complex, assembled at a centromere-like sequence. Consistent with previous work, we show that an ftsK-null mutant produces anucleate spores at the same frequency as the wild-type strain (0.8%). We report that the smc and ftsK deletion-insertion mutants (ftsK′ truncation allele) have developmental segregation defects (7% and 15% anucleate spores, respectively). By use of these latter mutants, viable double and triple mutants were isolated in all combinations with a previously described parB-null mutant (12% anucleate spores). parB and smc were in separate segregation pathways; the loss of both exacerbates the segregation defect (24% anucleate spores). For a triple mutant, deletion of the region encoding the FtsK motor domain and one transmembrane segment partially alleviates the segregation defect of the smc parB mutant (10% anucleate spores). Considerable redundancy must exist in this filamentous organism because segregation of some genomic material occurs 90% of the time during development in the absence of three functions with only a fourfold loss of spore viability. Furthermore, we report that scpA and scpAB mutants (encoding SMC-associated proteins) have spore nucleoid organization defects. Finally, FtsK-enhanced green fluorescent protein (EGFP) localized as bands or foci between incipient nucleoids, while SMC-EGFP foci were not uniformly positioned along aerial hyphae, nor were they associated with every condensing nucleoid.     

SigF, a new sigma factor required for a motility system of Myxococcus xanthus

A new sigma factor, SigF, was identified from the social and developmental bacterium Myxococcus xanthus. SigF is required for fruiting body formation during development as well as social motility during vegetative growth. Analysis of gene expression indicates that it is possible that the sigF gene is involved in regulation of an unidentified gene for social motility.     

High-Mobility-Group A-Like CarD Binds to a DNA Site Optimized for Affinity and Position and to RNA Polymerase To Regulate a Light-Inducible Promoter in Myxococcus xanthus

The CarD-CarG complex controls various cellular processes in the bacterium Myxococcus xanthus including fruiting body development and light-induced carotenogenesis. The CarD N-terminal domain, which defines the large CarD_CdnL_TRCF protein family, binds to CarG, a zinc-associated protein that does not bind DNA. The CarD C-terminal domain resembles eukaryotic high-mobility-group A (HMGA) proteins, and its DNA binding AT hooks specifically recognize the minor groove of appropriately spaced AT-rich tracts. Here, we investigate the determinants of the only known CarD binding site, the one crucial in CarD-CarG regulation of the promoter of the carQRS operon (P_QRS), a light-inducible promoter dependent on the extracytoplasmic function (ECF) σ factor CarQ. In vitro, mutating either of the 3-bp AT tracts of this CarD recognition site (TTTCCAGAGCTTT) impaired DNA binding, shifting the AT tracts relative to P_QRS had no effect or marginally lowered DNA binding, and replacing the native site by the HMGA1a binding one at the human beta interferon promoter (with longer AT tracts) markedly enhanced DNA binding. In vivo, however, all of these changes deterred P_QRS activation in wild-type M. xanthus, as well as in a strain with the CarD-CarG pair replaced by the Anaeromyxobacter dehalogenans CarD-CarG (CarD_Ad-CarG_Ad). CarD_Ad-CarG_Ad is functionally equivalent to CarD-CarG despite the lower DNA binding affinity in vitro of CarD_Ad, whose C-terminal domain resembles histone H1 rather than HMGA. We show that CarD physically associates with RNA polymerase (RNAP) specifically via interactions with the RNAP β subunit. Our findings suggest that CarD regulates a light-inducible, ECF σ-dependent promoter by coupling RNAP recruitment and binding to a specific DNA site optimized for affinity and position.