DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage

Summary The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 by consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.     

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.     

Initiation of chromosomal DNA synthesis in vitro

Summary An in vitro complementation assay for initiation of chromosomal DNA replication is described. The initiation reaction is dependent upon extract from either of two hybrid-plasmid containing strains. Each hybrid plasmid carries a suppressor of dnaA-ts mutations. The in vitro DNA synthesis is heavily biased toward the origin region, and the origin of replication (oriC) is replicated as determined by DNA-DNA hybridizations.     

The characterization of conserved binding motifs and potential target genes for <Emphasis Type="Italic">M. tuberculosis</Emphasis> MtrAB reveals a link between the two-component system and the drug resistance of <Emphasis Type="Italic">M. smegmatis</Emphasis>

Background  

The two-component systems of Mycobacterium tuberculosis are apparently required for its growth and resistance in hostile host environments. In such environments, MtrAB has been reported to regulate the expression of the M. tuberculosis replication initiator gene, dnaA. However, the dnaA promoter binding sites and many potential target genes for MtrA have yet to be precisely characterized.     

Unique organization of the dnaA region from Prochlorococcus marinus CCMP1375, a marine cyanobacterium

In order to study DNA replication control elements in cyanobacteria we cloned and sequenced the dnaA gene from the marine cyanobacterium Prochlorococcus marinus. The dnaA gene is ubiquitous among bacteria and encodes the DNA replication initiation factor DnaA. The deduced amino acid sequence of the P. marinus DnaA protein shows highest similarity to the DnaA protein from the freshwater cyanobacterium Synechocystis sp. PCC6803. Using a solid-phase DNA binding assay we demonstrated that both cyanobacterial DnaA proteins specifically recognize chromosomal origins, oriC, of Escherichia coli and Bacillus subtilis in vitro. The genetic environment of dnaA is not conserved between the two cyanobacteria. Upstream of the P. marinusdnaA gene we identified a gene encoding a putative ATP-binding cassette (ABC) transport protein. The gor gene encoding glutathione reductase lies downstream of dnaA. Comparison of the genetic structure of dnaA regions from 15 representative bacteria shows that the pattern of genes flanking dnaA is not universally conserved among them. Received: 20 July 1997 / Accepted: 7 October 1997     

Branched-chain fatty acids: the case for a novel form of cell-cell signalling during Myxococcus xanthus development

The esg locus is required for the formation of muiti-cellular fruiting bodies and spores by the developmental bacterium Myxococcus xanthus Studies have suggested that esg mutants are defective in the production of an essential signal (E-signal) used in cell-cell communication and that E-signalling is required for the expression of many developmental genes. Recently we have determined that the esg locus encodes components of a branched-chain keto acid dehydrogenase. a multienzyme complex involved in branched-chain amino acid metabolism in many bacteria and higher organisms. During vegetative growth in M. xanthus. this enzyme complex appears to participate in the production of the branched-chain fatty acids found in this organism. M. xanthus fatty acids (including the branched-chain fatty acids) have been observed to have a variety of effects on developing cells. These effects include; (i) the lysis of M. xanthus cells (autocide activity), (ii) acceleration of the rate of sporulation and (iii) rescue of sporulation by certain development-defective mutants. These and other results suggest a model in which the branched-chain fatty acids. Synthesized during growth, are released from cellular phospholipid by a developmentally regulated phospholipase during fruiting-body formation. This model proposes that one or more of the branched-chain fatty acids that are released constitutes the E-signal which must be transmitted between cells to complete M. xanthus development.     

Identification of the lipopolysaccharide O‐antigen biosynthesis priming enzyme and the O‐antigen ligase in Myxococcus xanthus: critical role of LPS O‐antigen in motility and development

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell–cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O‐antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O‐antigen in M. xanthus social behaviors. We identified WbaP_Mx (MXAN_2922) as the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for priming O‐antigen synthesis. In heterologous expression experiments, WbaP_Mx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O‐antigen synthesis, indicating that WbaP_Mx transfers galactose‐1‐P to undecaprenyl‐phosphate. We also identified WaaL_Mx (MXAN_2919), as the O‐antigen ligase that joins O‐antigen to lipid A‐core. Our data also support the previous suggestion that Wzm_Mx (MXAN_4622) and Wzt_Mx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O‐antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaP_Mx deletion mutant, we revisited the role of LPS O‐antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili‐dependent motility and required to complete the developmental program leading to the formation of spore‐filled fruiting bodies.     

Type IV‐pili dependent motility is co‐regulated by PilSR and PilS2R2 two‐component systems via distinct pathways in Myxococcus xanthus

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type‐IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P‐dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two‐component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P‐dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P‐dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P‐dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.     

Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in Escherichia coli is a DnaA-dependent process

Summary The thermosensitivity of dnaA(Ts) mutations can be suppressed by integration of plasmid F (integrative suppression). In the light of the recent finding that F requires DnaA protein for both establishment and maintenance, integrative suppression of 11 dnaA(Ts) mutations by a mini-F, pML31, integrated near oriC was examined. The plating efficiency of integratively suppressed strains was dnaA(Ts) allele-dependent and medium-dependent. The initiation capability of suppressed dnaA(Ts) strains lacking the oriC site and their F^- counterparts was determined at various temperatures between 30°C and 42°C. The degree of integrative suppression measured by the initiation capability varied in a dnaA(Ts) allele-dependent manner. F-directed DNA replication was most affected by the dnaA(Ts) mutations mapping in the middle of the gene whereas oriC-dependent replication was most thermosensitive in strains carrying mutations mapping in the carboxy-terminal half of the gene. The results indicated that the integrative suppression by F plasmid is a DnaA-dependent process and suggested that the requirements for DnaA protein in the oriC-dependent replication and F replication processes are qualitatively different.     

The effects of an Escherichia coli dnaAts mutation on the replication of the plasmids colE1 pSC101, R100.1 and RTF-TC.

Summary The rate of replication of the plasmids colE1, pSC101, R100.1 and pAR132 (an RTF-TC derivative of the drug resistance factor R100.1) has been investigated directly by DNA: DNA hybridization. These rates have been compared, in a dnaAts strain, to that of various markers of the host chromosome at permissive and non-permissive temperatures. Chromosome initiation in the dnaAts strain stops rapidly after a shift to the non-permissive temperature, but plasmids R100.1 and pAR132 do not seem to be affected directly and continue replication for some time. The colE1 replication rate undergoes a large increase after the temperature shift, followed by a rapid decrease to a very low level 25 min after the shift. In contrast pSC101 replication stops immediately after the shift. ColE1 is able to replicate in an integratively suppressed dnaAts strain at 42° C whereas pSC101 stops replication immediately under these conditions. We conclude that R100.1 and its derivative RTF-TC can replicate without a functional dnaA product; that colE1, while affected by a shift in temperature in a dnaAts strain, does not directly require dnaA; and that the plasmid pSC101 has an absolute requirement for dnaA. The absolute requirement of pSC101 for dnaA in the integratively suppressed Hfr strain provides a useful system for further investigations of the dnaA function.     

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.     

Genetics of gliding motility and development inMyxococcus xanthus

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryoteMyxococcus xanthus. As part of its life cycle,M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential forM. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development inM. xanthus is induced by starvation, and early events in development suggest that signaling, stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, α-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding, motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development inM. xanthus.     

Replication of a Bacillus subtilis oriC plasmid in vitro

We constructed an in vitro replication system specific for a Bacillus subtilis oriC plasmid using a soluble fraction derived from cell extracts of B. subtilis. DNA polymerase III and two initiation proteins, DnaA and DnaB, were required for in vitro replication as observed in vivo. Both upstream and downstream DnaA box regions of the dnaA gene were required as cis-elements for in vitro synthesis of the B. subtilis oriC plasmid as well as for in vivo activity. The replication was semi-conservative and only one round of replication occurred within 15min. These results indicate that in vitro replication faithfully reproduced in vivo replication. To elucidate the site of initiation and the direction of replication, we analysed replicative intermediates generated in vitro in the presence of various concentrations of ddGTP by two methods. First, analysis of restriction fragments around the dnaA gene showed a high level of incorporation of the radioactive substrate, indicating that replication began within the vicinity of the dnaA gene. Second, using 2-dimensional gel electrophoresis, bubble arcs were detected only on fragments containing the DnaA box region downstream of the dnaA gene, indicating that the initiation site resided within this region. The distribution of the bubble arcs suggested that both bidirectional and unidirectional replication occurred in vitro.     

Continuous synthesis of the dnaA gene product of Escherichia coli in the cell cycle

Summary The dnaA gene product of Escherichia coli, identified as a weakly basic protein of about 48,000 daltons (Yuasa and Sakakibara 1980), can be separated from other celluar proteins by means of two-dimensional gel electrophoresis. Synthesis of the dnaA protein took place continuously during a cell growth cycle. The newly synthesized dnaA protein persisted stably for one generation. Thermosensitive dnaA protein produced by the dnaA167 mutant was stable at 30° C, but was disintegrated at 42° C. The amount of intact dnaA protein present in the mutant exposed to the high temperature for 60 min was less than a quarter of the amount at the time of the shift. The cells having the reduced amount of intact dnaA protein were capable of initiating a new round of chromosome replication at the low temperature without de novo synthesis of the dnaA protein. The potential of the mutant for initiation of DNA replication decreased with reduction in the amount of the thermoreversible dnaA protein. The mutations dnaA167 and dnaA46 had no significant effect on the syntheses of the dnaA mRNA and the protein product at the low and high temperatures.Abbreviations used SDS sodium dodecyl sulfate - kb kilobase pairs - TCA trichloroacetic acid     

Glycine Betaine Biosynthesized from Glycine Provides an Osmolyte for Cell Growth and Spore Germination during Osmotic Stress in Myxococcus xanthus

Glycine sarcosine methyltransferase (Gsm) and sarcosine dimethylglycine methyltransferase (Sdm) catalyze glycine betaine synthesis from glycine. Disruption of the M. xanthus gsmA (MXAN 7068) or sdmA (MXAN 3190) gene, encoding Gsm or Sdm homologue proteins, respectively, generated mutants that exhibited a longer lag period of growth and delayed spore germination under osmostress.Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex multicellular developmental cycle (6, 7). These bacteria live in soil, where they prey on other microbes for food. In response to nutritional stress, hundreds of thousands of vegetative cells aggregate to form multicellular fruiting bodies containing differentiated myxospores. Once conditions become favorable for growth, the desiccation- and heat-resistant spores can germinate and initiate vegetative growth.It was reported previously that the receptor-type adenylyl cyclases CyaA and CyaB of M. xanthus act as osmosensors during spore germination and growth, respectively (8, 9). Glycine betaine is a very efficient osmolyte found in a wide range of prokaryotic and eukaryotic organisms, where it is accumulated at high cytoplasmic concentrations in response to osmotic stress (4, 16). In this study, it is reported that in M. xanthus glycine betaine can be biosynthesized from glycine and mainly functions as an osmoprotectant for cell growth and spore germination under osmotic stress conditions.