The DNA damage response of Escherichia coli, revisited: Differential gene expression after replication inhibition.

In 1967, in this journal, Evelyn Witkin proposed the existence of a coordinated DNA damage response in Escherichia coli, which later came to be called the "SOS response." We revisited this response using the replication inhibitor azidothymidine (AZT) and RNA-Seq analysis and identified several features. We confirm the induction of classic Save our ship (SOS) loci and identify several genes, including many of the pyrimidine pathway, that have not been previously demonstrated to be DNA damage-inducible. Despite a strong dependence on LexA, these genes lack LexA boxes and their regulation by LexA is likely to be indirect via unknown factors. We show that the transcription factor "stringent starvation protein" SspA is as important as LexA in the regulation of AZT-induced genes and that the genes activated by SspA change dramatically after AZT exposure. Our experiments identify additional LexA-independent DNA damage inducible genes, including 22 small RNA genes, some of which appear to activated by SspA. Motility and chemotaxis genes are strongly down-regulated by AZT, possibly as a result of one of more of the small RNAs or other transcription factors such as AppY and GadE, whose expression is elevated by AZT. Genes controlling the iron siderophore, enterobactin, and iron homeostasis are also strongly induced, independent of LexA. We confirm that IraD antiadaptor protein is induced independent of LexA and that a second antiadaptor, IraM is likewise strongly AZT-inducible, independent of LexA, suggesting that RpoS stabilization via these antiadaptor proteins is an integral part of replication stress tolerance.

Structural basis for regulation of SOS response in bacteria.

In response to DNA damage, bacterial RecA protein forms filaments with the assistance of DinI protein. The RecA filaments stimulate the autocleavage of LexA, the repressor of more than 50 SOS genes, and activate the SOS response. During the late phase of SOS response, the RecA filaments stimulate the autocleavage of UmuD and λ repressor CI, leading to mutagenic repair and lytic cycle, respectively. Here, we determined the cryo-electron microscopy structures of Escherichia coli RecA filaments in complex with DinI, LexA, UmuD, and λCI by helical reconstruction. The structures reveal that LexA and UmuD dimers bind in the filament groove and cleave in an intramolecular and an intermolecular manner, respectively, while λCI binds deeply in the filament groove as a monomer. Despite their distinct folds and oligomeric states, all RecA filament binders recognize the same conserved protein features in the filament groove. The SOS response in bacteria can lead to mutagenesis and antimicrobial resistance, and our study paves the way for rational drug design targeting the bacterial SOS response.

Anti-mutagenic agent targeting LexA to combat antimicrobial resistance in mycobacteria.

Antimicrobial resistance (AMR) is a serious global threat demanding innovations for effective control of pathogens. The bacterial SOS response, regulated by the master regulators, LexA and RecA, contributes to AMR through advantageous mutations. Targeting the LexA/RecA system with a novel inhibitor could suppress the SOS response and potentially reduce the occurrence of AMR. RecA presents a challenge as a therapeutic target due to its conserved structure and function across species, including humans. Conversely, LexA which is absent in eukaryotes, can be potentially targeted, due to its involvement in SOS response which is majorly responsible for adaptive mutagenesis and AMR. Our studies combining bioinformatic, biochemical, biophysical, molecular, and cell-based assays present a unique inhibitor of mycobacterial LexA, wherein we show that the inhibitor interacts directly with the catalytic site residues of LexA of Mycobacterium tuberculosis (Mtb), consequently hindering its cleavage, suppressing SOS response thereby reducing mutation frequency and AMR.

Repression of Acinetobacter baumannii DNA damage response requires DdrR-assisted binding of UmuDAb dimers to atypical SOS box.

The DNA damage response of the multi-drug-resistant nosocomial pathogen Acinetobacter baumannii possesses multiple features that distinguish it from the commonly used LexA repression system. These include the absence of LexA in this genus, the evolution of a UmuD polymerase manager into the UmuDAb repressor of error-prone polymerases, the use of a corepressor unique to Acinetobacter (DdrR), and an unusually large UmuDAb binding site. We defined cis- and trans-acting factors required for UmuDAb DNA binding and gene repression, and tested whether DdrR directly enhances its DNA binding. We used DNA binding assays to characterize UmuDAb's binding to its proposed operator present upstream of the six co-repressed umuDC or umuC genes. UmuDAb bound tightly and cooperatively to this site with ~10-fold less affinity than LexA. DdrR enhanced the binding of both native and dimerization-deficient UmuDAb forms, but only in greater than equimolar ratios relative to UmuDAb. UmuDAb mutants unable to dimerize or effect gene repression showed impaired DNA binding, and a strain expressing the G124D dimerization mutant could not repress transcription of the UmuDAb-DdrR regulon. Competition electrophoretic mobility shift assays conducted with mutated operator probes showed that, unlike typical SOS boxes, the UmuDAb operator possessed a five-base pair central core whose sequence was more crucial for binding than the flanking palindrome. The presence of only one of the two flanking arms of the palindrome was necessary for UmuDAb binding. Overall, the data supported a model of an operator with two UmuDAb binding sites. The distinct characteristics of UmuDAb and its regulated promoters differ from the typical LexA repression model, demonstrating a novel method of repression.IMPORTANCEAcinetobacter baumannii is a gram-negative bacterium responsible for hospital-acquired infections. Its unique DNA damage response can activate multiple error-prone polymerase genes, allowing it to gain mutations that can increase its virulence and antibiotic resistance. The emergence of infectious strains carrying multiple antibiotic resistance genes, including carbapenem resistance, lends urgency to discovering and developing ways to combat infections resistant to treatment with known antibiotics. Deciphering how the regulators UmuDAb and DdrR repress the error-prone polymerases could lead to developing complementary treatments to halt this mechanism of generating resistance.

Strain-Specific Gifsy-1 Prophage Genes Are Determinants for Expression of the RNA Repair Operon during the SOS Response in Salmonella enterica Serovar Typhimurium.

The adaptation of Salmonella enterica serovar Typhimurium to stress conditions involves expression of genes within the regulon of the alternative sigma factor RpoN (σ54). RpoN-dependent transcription requires an activated bacterial enhancer binding protein (bEBP) that hydrolyzes ATP to remodel the RpoN-holoenzyme-promoter complex for transcription initiation. The bEBP RtcR in S. Typhimurium strain 14028s is activated by genotoxic stress to direct RpoN-dependent expression of the RNA repair operon rsr-yrlBA-rtcBA. The molecular signal for RtcR activation is an oligoribonucleotide with a 3'-terminal 2',3'-cyclic phosphate. We show in S. Typhimurium 14028s that the molecular signal is not a direct product of nucleic acid damage, but signal generation is dependent on a RecA-controlled SOS-response pathway, specifically, induction of prophage Gifsy-1. A genome-wide mutant screen and utilization of Gifsy prophage-cured strains indicated that the nucleoid-associated protein Fis and the Gifsy-1 prophage significantly impact RtcR activation. Directed-deletion analysis and genetic mapping by transduction demonstrated that a three-gene region (STM14_3218-3220) in Gifsy-1, which is variable between S. Typhimurium strains, is required for RtcR activation in strain 14028s and that the absence of STM14_3218-3220 in the Gifsy-1 prophages of S. Typhimurium strains LT2 and 4/74, which renders these strains unable to activate RtcR during genotoxic stress, can be rescued by complementation in cis by the region encompassing STM14_3218-3220. Thus, even though RtcR and the RNA repair operon are highly conserved in Salmonella enterica serovars, RtcR-dependent expression of the RNA repair operon in S. Typhimurium is controlled by a variable region of a prophage present in only some strains. IMPORTANCE The transcriptional activator RtcR and the RNA repair proteins whose expression it regulates, RtcA and RtcB, are widely conserved in Proteobacteria. In Salmonella Typhimurium 14028s, genotoxic stress activates RtcR to direct RpoN-dependent expression of the rsr-yrlBA-rtcBA operon. This work identifies key elements of a RecA-dependent pathway that generates the signal for RtcR activation in strain 14028s. This signaling pathway requires the presence of a specific region within the prophage Gifsy-1, yet this region is absent in most other wild-type Salmonella strains. Thus, we show that the activity of a widely conserved regulatory protein can be controlled by prophages with narrow phylogenetic distributions. This work highlights an underappreciated phenomenon where bacterial physiological functions are altered due to genetic rearrangement of prophages.

Regulation of phosphate starvation-specific responses in Escherichia coli.

Toxic agents added into the medium of rapidly growing Escherichia coli induce specific stress responses through the activation of specialized transcription factors. Each transcription factor and downstream regulon (e.g. SoxR) are linked to a unique stress (e.g. superoxide stress). Cells starved of phosphate induce several specific stress regulons during the transition to stationary phase when the growth rate is steadily declining. Whereas the regulatory cascades leading to the expression of specific stress regulons are well known in rapidly growing cells stressed by toxic products, they are poorly understood in cells starved of phosphate. The intent of this review is to both describe the unique mechanisms of activation of specialized transcription factors and discuss signalling cascades leading to the induction of specific stress regulons in phosphate-starved cells. Finally, I discuss unique defence mechanisms that could be induced in cells starved of ammonium and glucose.

Genome-Wide Identification of the LexA-Mediated DNA Damage Response in Streptomyces venezuelae.

DNA damage triggers a widely conserved stress response in bacteria called the SOS response, which involves two key regulators, the activator RecA and the transcriptional repressor LexA. Despite the wide conservation of the SOS response, the number of genes controlled by LexA varies considerably between different organisms. The filamentous soil-dwelling bacteria of the genus Streptomyces contain LexA and RecA homologs, but their roles in Streptomyces have not been systematically studied. Here, we demonstrate that RecA and LexA are required for the survival of Streptomyces venezuelae during DNA-damaging conditions and for normal development during unperturbed growth. Monitoring the activity of a fluorescent recA promoter fusion and LexA protein levels revealed that the activation of the SOS response is delayed in S. venezuelae. By combining global transcriptional profiling and chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we determined the LexA regulon and defined the core set of DNA damage repair genes that are expressed in response to treatment with the DNA-alkylating agent mitomycin C. Our results show that DNA damage-induced degradation of LexA results in the differential regulation of LexA target genes. Using surface plasmon resonance, we further confirmed the LexA DNA binding motif (SOS box) and demonstrated that LexA displays tight but distinct binding affinities to its target promoters, indicating a graded response to DNA damage. IMPORTANCE The transcriptional regulator LexA functions as a repressor of the bacterial SOS response, which is induced under DNA-damaging conditions. This results in the expression of genes important for survival and adaptation. Here, we report the regulatory network controlled by LexA in the filamentous antibiotic-producing Streptomyces bacteria and establish the existence of the SOS response in Streptomyces. Collectively, our work reveals significant insights into the DNA damage response in Streptomyces that will promote further studies to understand how these important bacteria adapt to their environment.

The Small DdrR Protein Directly Interacts with the UmuDAb Regulator of the Mutagenic DNA Damage Response in Acinetobacter baumannii.

Acinetobacter baumannii poses a great threat in health care settings worldwide, with clinical isolates displaying an ever-evolving multidrug resistance. In strains of A. baumannii, expression of multiple error-prone polymerase genes are corepressed by UmuDAb, a member of the LexA superfamily, and a small protein, DdrR. It is currently unknown how DdrR establishes this repression. Here, we used surface plasmon resonance spectrometry to show that DdrR formed a stable complex with the UmuDAb regulator. Our results indicated that the carboxy-terminal dimerization domain of UmuDAb formed the interaction interface with DdrR. Our in vitro data also showed that RecA-mediated inactivation of UmuDAb was inhibited when this transcription factor was bound to its target DNA. In addition, we showed that DdrR interacted with a putative prophage repressor, homologous to LexA superfamily proteins. These data suggested that DdrR modulated DNA damage response and prophage induction in A. baumannii by binding to LexA-like regulators. IMPORTANCE We previously identified a 50-residue bacteriophage protein, gp7, which interacts with and modulates the function of the LexA transcription factor from Bacillus thuringiensis. Here, we present data that indicates that the small DdrR protein from A. baumannii likely coordinates the SOS response and prophage processes by also interacting with LexA superfamily members. We suggest that similar small proteins that interact with LexA-like proteins to coordinate DNA repair and bacteriophage functions may be common to many bacteria that mount the SOS response.

Expression and Characterization of the Staphylococcus aureus RecA protein: A mapping of canonical functions.

Recombinases are responsible for homologous recombination (HR), proper genome maintenance, and accurate deoxyribonucleic acid (DNA) duplication. Moreover, HR plays a determining role in DNA transaction processes such as DNA replication, repair, recombination, and transcription. Staphylococcus aureus, an opportunistic pathogen, usually causes respiratory infections such as sinusitis, skin infections, and food poisoning. To date, the role of the RecA gene product in S. aureus remains obscure. In this study, we attempted to map the functional properties of the RecA protein. S. aureus expresses the recA gene product in vivo upon exposure to the DNA-damaging agents, ultraviolet radiation, and methyl methanesulfonate. The recombinant purified S. aureus RecA protein displayed strong single-stranded DNA affinity compared to feeble binding to double-stranded DNA. Interestingly, the RecA protein is capable of invasion and formed displacement loops and readily performed strand-exchange activities with an oligonucleotide-based substrate. Notably, the S. aureus RecA protein hydrolyzed the DNA-dependent adenosine triphosphate and cleaved LexA, showing the conserved function of coprotease. This study provides the functional characterization of the S. aureus RecA protein and sheds light on the canonical processes of homologous recombination, which are conserved in the gram-positive foodborne pathogen S. aureus.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

Two components of DNA replication-dependent LexA cleavage.

Induction of the SOS response, a cellular system triggered by DNA damage in bacteria, depends on DNA replication for the generation of the SOS signal, ssDNA. RecA binds to ssDNA, forming filaments that stimulate proteolytic cleavage of the LexA transcriptional repressor, allowing expression of > 40 gene products involved in DNA repair and cell cycle regulation. Here, using a DNA replication system reconstituted in vitro in tandem with a LexA cleavage assay, we studied LexA cleavage during DNA replication of both undamaged and base-damaged templates. Only a ssDNA-RecA filament supported LexA cleavage. Surprisingly, replication of an undamaged template supported levels of LexA cleavage like that induced by a template carrying two site-specific cyclobutane pyrimidine dimers. We found that two processes generate ssDNA that could support LexA cleavage. 1) During unperturbed replication, single-stranded regions formed because of stochastic uncoupling of the leading-strand DNA polymerase from the replication fork DNA helicase, and 2) on the damaged template, nascent leading-strand gaps were generated by replisome lesion skipping. The two pathways differed in that RecF stimulated LexA cleavage during replication of the damaged template, but not normal replication. RecF appears to facilitate RecA filament formation on the leading-strand ssDNA gaps generated by replisome lesion skipping.

The PafBC-mediated response sensitizes a bistable DNA damage response in Mycobacteria.

Due to the genotoxically challenging environments in which they live in, Mycobacteria have a complex DNA damage repair system that is governed by two major DNA damage responses, namely, the LexA/RecA-dependent response and the newly characterized PafBC-mediated response (Müller et al., 2018). The LexA/RecA-dependent response is a well-known bistable response found in different types of bacteria, and the Mycobacteria-specific PafBC-mediated response interacts with and modifies the LexA/RecA-dependent response (Müller et al., 2018). The interaction between the LexA/RecA-dependent response and the PafBC-mediated response has not been characterized mathematically. Our analysis shows that the addition of the PafBC-mediated response sensitizes the overall DNA damage response, effectively lowering the DNA damage rate threshold for activation.

Homodimerization and heterodimerization requirements of Acinetobacter baumannii SOS response coregulators UmuDAb and DdrR revealed by two-hybrid analyses.

The multidrug-resistant pathogen Acinetobacter baumannii displays unusual control of its SOS mutagenesis genes, as it does not encode a LexA repressor, but instead employs the UmuDAb repressor and a small protein, DdrR, that is uniquely found in Acinetobacter species. We used bacterial adenylate cyclase two-hybrid analyses to determine if UmuDAb and DdrR coregulation might involve physical interactions. Neither quantitative nor qualitative assays showed UmuDAb interaction with DdrR. DdrR hybrid proteins, however, demonstrated modest head-to-tail interactions in a qualitative assay. The similarity of UmuDAb to the homodimer-forming polymerase manager UmuD and LexA repressor proteins suggested that it may form dimers, which we observed. UmuDAb homodimerization required a free C terminus, and either small truncations or addition of a histidine tag at the C terminus abolished this homodimerization. The amino acid N100, crucial for UmuD dimer formation, was dispensable if both C termini were free to interact. However, mutation of the amino acid G124, necessary for LexA dimerization, yielded significantly less UmuDAb dimerization, even if both C termini were free. This suggests that UmuDAb forms dimers like LexA does, but may not coregulate gene expression involving a physical association with DdrR. The homodimerization of these coregulators provides insight into a LexA-independent, coregulatory process of controlling a conserved bacterial action such as the mutagenic DNA damage response.

A corepressor participates in LexA-independent regulation of error-prone polymerases in Acinetobacter.

The DNA damage response of the multidrug-resistant pathogen Acinetobacter baumannii, which induces mutagenic UmuD'2C error-prone polymerases, differs from that of many bacteria. Acinetobacter species lack a LexA repressor, but induce gene transcription after DNA damage. One regulator, UmuDAb, binds to and represses the promoters of the multiple A. baumannii ATCC 17978 umuDC alleles and the divergently transcribed umuDAb and ddrR genes. ddrR is unique to the genus Acinetobacter and of unknown function. 5' RACE (rapid amplification of cDNA ends) PCR mapping of the umuDAb and ddrR transcriptional start sites revealed that their -35 promoter elements overlapped the UmuDAb binding site, suggesting that UmuDAb simultaneously repressed expression of both genes by blocking polymerase access. This coordinated control of ddrR and umuDAb suggested that ddrR might also regulate DNA damage-inducible gene transcription. RNA-sequencing experiments in 17 978 ddrR- cells showed that ddrR regulated approximately 25 % (n=39) of the mitomycin C-induced regulon, with umuDAb coregulating 17 of these ddrR-regulated genes. Eight genes (the umuDC polymerases, umuDAb and ddrR) were de-repressed in the absence of DNA damage, and nine genes were uninduced in the presence of DNA damage, in both ddrR and umuDAb mutant strains. These data suggest ddrR has multiple roles, both as a co-repressor and as a positive regulator of DNA damage-inducible gene transcription. Additionally, 57 genes were induced by mitomycin C in the ddrR mutant but not in wild-type cells. This regulon contained multiple genes for DNA replication, recombination and repair, transcriptional regulators, RND efflux, and transport. This study uncovered another regulator of the atypical DNA damage response of this genus, to help describe how this pathogen acquires drug resistance through its expression of the error-prone polymerases under DdrR and UmuDAb control.

Ordering up gene expression by slowing down transcription factor binding kinetics.

Efficient regulation of a complex genetic response requires that the gene products, which catalyze the response, be synthesized in a temporally ordered manner to match the sequential nature of the reaction pathway they act upon. Transcription regulation networks coordinate this aspect of cellular control by modulating transcription factor (TF) concentrations through time. The effect a TF has on the timing of gene expression is often modeled assuming that the TF-promoter binding reaction is in thermodynamic equilibrium with changes in TF concentration over time; however, non-equilibrium dynamics resulting from relatively slow TF-binding kinetics can result in different network behavior. Here, I highlight a recent study of the bacterial SOS response, where a single TF regulates multiple target promoters, to show how a disequilibrium of TF binding at promoters results in a more complex behavior, enabling a larger temporal separation of promoter activities that depends not only upon slow TF binding kinetics at promoters, but also on the magnitude of the response stimulus. I also discuss the dependence of network behavior on specific TF regulatory mechanisms and the implications non-equilibrium dynamics have for stochastic gene expression.

Differential efficacy of genetically swapping GAL4.

Several large or mid-scale collections of Drosophila enhancer traps have been recently created to allow for genetic swapping of GAL4 coding sequences to versatile transcription activators or suppressors such as LexA, QF, split-GAL4 (GAL4-AD and GAL4-DBD), GAL80 and QS. Yet a systematic analysis of the feasibility and reproducibility of these tools is lacking. Here we focused on InSITE GAL4 drivers that specifically label different subpopulations of olfactory neurons, particularly local interneurons (LNs), and genetically swapped the GAL4 domain for LexA, GAL80 or QF at the same locus. We found that the major utility-limiting factor for these genetic swaps is that many do not fully reproduce the original GAL4 expression patterns. Different donors exhibit distinct efficacies for reproducing original GAL4 expression patterns. The successfully swapped lines reported here will serve as valuable reagents and expand the genetic toolkits of Drosophila olfactory circuit research.

Unique patterns of organization and migration of FGF-expressing cells during Drosophila morphogenesis.

Fibroblast growth factors (FGF) are essential signaling proteins that regulate diverse cellular functions in developmental and metabolic processes. In Drosophila, the FGF homolog, branchless (bnl) is expressed in a dynamic and spatiotemporally restricted pattern to induce branching morphogenesis of the trachea, which expresses the Bnl-receptor, breathless (btl). Here we have developed a new strategy to determine bnl- expressing cells and study their interactions with the btl-expressing cells in the range of tissue patterning during Drosophila development. To enable targeted gene expression specifically in the bnl expressing cells, a new LexA based bnl enhancer trap line was generated using CRISPR/Cas9 based genome editing. Analyses of the spatiotemporal expression of the reporter in various embryonic stages, larval or adult tissues and in metabolic hypoxia, confirmed its target specificity and versatility. With this tool, new bnl expressing cells, their unique organization and functional interactions with the btl-expressing cells were uncovered in a larval tracheoblast niche in the leg imaginal discs, in larval photoreceptors of the developing retina, and in the embryonic central nervous system. The targeted expression system also facilitated live imaging of simultaneously labeled Bnl sources and tracheal cells, which revealed a unique morphogenetic movement of the embryonic bnl- source. Migration of bnl- expressing cells may create a dynamic spatiotemporal pattern of the signal source necessary for the directional growth of the tracheal branch. The genetic tool and the comprehensive profile of expression, organization, and activity of various types of bnl-expressing cells described in this study provided us with an important foundation for future research investigating the mechanisms underlying Bnl signaling in tissue morphogenesis.

The LexA transcription factor regulates fatty acid biosynthetic genes in the cyanobacterium Synechocystis sp. PCC 6803.

Specific transcription factors have been identified in various heterotrophic bacterial species that regulate the sets of genes required for fatty acid metabolism. Here, we report that expression of the fab genes, encoding fatty acid biosynthetic enzymes, is regulated by the global regulator LexA in the photoautotrophic cyanobacterium Synechocystis sp. PCC 6803. Sll1626, an ortholog of the well-known LexA repressor involved in the SOS response in heterotrophic bacteria, was isolated from crude extracts of Synechocystis by DNA affinity chromatography, reflecting its binding to the upstream region of the acpP-fabF and fabI genes. An electrophoresis mobility shift assay revealed that the recombinant LexA protein can bind to the upstream region of each fab gene tested (fabD, fabH, fabF, fabG, fabZ and fabI). Quantitative RT-PCR analysis of the wild type and a lexA-disrupted mutant strain suggested that LexA acts as a repressor of the fab genes involved in initiation of fatty acid biosynthesis (fabD, fabH and fabF) and the first reductive step in the subsequent elongation cycle (fabG) under normal growth conditions. Under nitrogen-depleted conditions, downregulation of fab gene expression is partly achieved through an increase in LexA-repressing activity. In contrast, under phosphate-depleted conditions, fab gene expression is upregulated, probably due to the loss of repression by LexA. We further demonstrate that elimination of LexA largely increases the production of fatty acids in strains modified to secrete free fatty acids.

Novel genetic approaches to behavior in Drosophila.

The study of behavior requires manipulation of the controlling neural circuits. The fruit fly, Drosophila melanogaster, is an ideal model for studying behavior because of its relatively small brain and the numerous sophisticated genetic tools that have been developed for this animal. Relatively recent technical advances allow the manipulation of a small subset of neurons with temporal resolution in flies while they are subject to behavior assays. This review briefly describes the most important genetic techniques, reagents, and approaches that are available to study and manipulate the neural circuits involved in Drosophila behavior. We also describe some examples of these genetic tools in the study of the olfactory receptor system.

Differential regulation of ssb genes in the nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC71201.

Anabaena sp. PCC7120 possesses three genes coding for single-stranded DNA-binding (SSB) protein, of which ssb1 was a single gene, and ssb2 and ssb3 are the first genes of their corresponding operons. Regulation of the truncated ssb genes, ssb1 (alr0088) and ssb2 (alr7559), was unaffected by N-status of growth. They were negatively regulated by the SOS-response regulatory protein LexA, as indicated by the (i) binding of Anabaena LexA to the LexA box of regulatory regions of ssb1 and ssb2, and (ii) decreased expression of the downstream gfp reporter gene in Escherichia coli upon co-expression of LexA. However, the full-length ssb gene, ssb3 (all4779), was regulated by the availability of Fe2+ and combined nitrogen, as indicated by (i) increase in the levels of SSB3 protein on Fe2+ -depletion and decrease under Fe2+ -excess conditions, and (ii) 1.5- to 1.6-fold decrease in activity under nitrogen-fixing conditions compared to nitrogen-supplemented conditions. The requirement of Fe2+ as a co-factor for repression by FurA and the increase in levels of FurA under nitrogen-deficient conditions in Anabaena (Lopez-Gomollon et al. 2007) indicated a possible regulation of ssb3 by FurA. This was substantiated by (i) the binding of FurA to the regulatory region of ssb3, (ii) repression of the expression of the downstream gfp reporter gene in E. coli upon co-expression of FurA, and (iii) negative regulation of ssb3 promoter activity by the upstream AT-rich region in Anabaena. This is the first report on possible role of FurA, an important protein for iron homeostasis, in DNA repair of cyanobacteria.