Imperfect Repeats Research Articles

Regulation of curcumin reductase curA (PA2197) through sodium hypochlorite and N-ethylmaleimide sensing by TetR family repressor CurR (PA2196) in Pseudomonas aeruginosa

Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5′-TAGTTGA-C-TGGTCTA-3′. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin.

Functional Amyloids: The Biomaterials of Tomorrow?

Functional amyloid (FAs), particularly the bacterial proteins CsgA and FapC, have many useful properties as biomaterials: high stability, efficient, and controllable formation of a single type of amyloid, easy availability as extracellular material in bacterial biofilm and flexible engineering to introduce new properties. CsgA in particular has already demonstrated its worth in hydrogels for stable gastrointestinal colonization and regenerative tissue engineering, cell-specific drug release, water-purification filters, and different biosensors. It also holds promise as catalytic amyloid; existing weak and unspecific activity can undoubtedly be improved by targeted engineering and benefit from the repetitive display of active sites on a surface. Unfortunately, FapC remains largely unexplored and no application is described so far. Since FapC shares many common features with CsgA, this opens the window to its development as a functional scaffold. The multiple imperfect repeats in CsgA and FapC form a platform to introduce novel properties, e.g., in connecting linkers of variable lengths. While exploitation of this potential is still at an early stage, particularly for FapC, a thorough understanding of their molecular properties will pave the way for multifunctional fibrils which can contribute toward solving many different societal challenges, ranging from CO2 fixation to hydrolysis of plastic nanoparticles.

Natural transformants of Camellia section Thea

Horizontal gene transfer (HGT) plays an important role in plant evolution and plant development. Agrobacterium-mediated gene transfer leads to the formation of crown galls or hairy roots, due to expression of transferred T-DNA genes. Spontaneous regeneration of transformed cells can produce natural transformants carrying cellular T-DNA (cT-DNA) sequences of bacterial origin. HGT from Agrobacterium to dicots is remarkably widespread. The production of naturally genome modified plants could play a role in plant evolution and environment.
 Among these natural GMOs (nGMOs) there are the tea plants. Camellia sinensis var. sinensis cv. Shuchazao contains a single 5.5 kb cT-DNA fragment organized as imperfect inverted repeat with three inactive genes. 142 Camellia accessions, belonging to 10 of 11 species of the section Thea, were studied for the presence of cT-DNA alleles. All of them contain the cT-DNA insert, indicating that they are resulted from the single transformed event. Allele phasing showed that 82 accessions were heterozygous for T-DNA alleles, 60 others were homozygous. A phylogenetic analysis of all found alleles showed existence of two separate groups of them, further divided into subgroups. The alleles of the different Camellia species were distributed mosaically over groups, and different species showed very similar T-DNA alleles. This indicates that the taxonomy of Thea requires revision. The nucleotide divergence of the imperfect cT-DNA repeats indicates that the age of cT-DNA insertion is about 15 mya, which is earlier then emergence of section Thea [1]. We present a working model for the origin and evolution of nGMO plants derived from allogamous transformants.

Location deviation correction method based oncross-correlation spectrum in OFDR.

A location deviation compensation algorithm based on cross-correlation spectrum is proposed for optical frequency domain reflection (OFDR) measurement. The strain and the imperfect repeatability of the laser source may cause a location deviation of the OFDR measurement system, resulting in a demodulation error. This paper proposes to use the peak-to-average ratio (PAR) to measure the quality of the correlation results and achieve compensation, which achieves efficient compensation for location deviation. Compared to other compensation methods, this algorithm can deal with the complex location deviation with better demodulation accuracy and stability. Through a strain measurement experiment, we demonstrate that the demodulation error caused by location deviation is largely eliminated. As a result, the spatial resolution of the system is improved from 8cm to 2cm, and the maximum measurement range is expanded to 5000µε.

Molecular characterization of the N-terminal half of TasA during amyloid-like assembly and its contribution to Bacillus subtilis biofilm formation

Biofilms are bacterial communities that result from a cell differentiation process leading to the secretion of an extracellular matrix (ECM) by part of the population. In Bacillus subtilis, the main protein component of the ECM is TasA, which forms a fiber-based scaffold that confers structure to the ECM. The N-terminal half of TasA is strongly conserved among Bacillus species and contains a protein domain, the rigid core (RcTasA), which is critical for the structural and functional properties of the recombinant protein. In this study, we demonstrate that recombinantly purified RcTasA in vitro retains biochemical properties previously observed for the entire protein. Further analysis of the RcTasA amino acid sequence revealed two aggregation-prone stretches and a region of imperfect amino acid repeats, which are known to contribute to functional amyloid assembly. Biochemical characterization of these stretches found in RcTasA revealed their amyloid-like capacity in vitro, contributing to the amyloid nature of RcTasA. Moreover, the study of the imperfect amino acid repeats revealed the critical role of residues D64, K68 and D69 in the structural function of TasA. Experiments with versions of TasA carrying the substitutions D64A and K68AD69A demonstrated a partial loss of function of the protein either in the assembly of the ECM or in the stability of the core and amyloid-like properties. Taken together, our findings allow us to better understand the polymerization process of TasA during biofilm formation and provide knowledge into the sequence determinants that promote the molecular behavior of protein filaments in bacteria.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5′ untranslated region (5′UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.

Dissecting the Self-assembly Dynamics of Imperfect Repeats in α-Synuclein.

The pathological aggregation of α-synuclein (αS) into amyloid fibrils is the hallmark of Parkinson's disease (PD). The self-assembly and membrane interactions of αS are mainly governed by the seven imperfect 11-residue repeats of the XKTKEGVXXXX motif around residues 1-95. However, the particular role of each repeat in αS fibrillization remains unclear. To answer this question, we studied the aggregation dynamics of each repeat with up to 10 peptides in silico by conducting multiple independent micro-second atomistic discrete molecular dynamics simulations. Our simulations revealed that only repeats R3 and R6 readily self-assembled into β-sheet-rich oligomers, while the other repeats remained as unstructured monomers with weak self-assembly and β-sheet propensities. The self-assembly process of R3 featured frequent conformational changes with β-sheet formation mainly in the non-conserved hydrophobic tail, whereas R6 spontaneously self-assembled into extended and stable cross-β structures. These results of seven repeats are consistent with their structures and organization in recently solved αS fibrils. As the primary amyloidogenic core, R6 was buried inside the central cross-β core of all αS fibrils, attracting the hydrophobic tails of adjacent R4, R5, and R7 repeats forming β-sheets around R6 in the core. Further away from R6 in the sequence but with a moderate amyloid aggregation propensity, the R3 tail could serve as a secondary amyloidogenic core and form independent β-sheets in the fibril. Overall, our results demonstrate the critical role of R3 and R6 repeats in αS amyloid aggregation and suggest their potential as targets for the peptide-based and small-molecule amyloid inhibitors.

ICESpsuAH0906, a novel optrA-carrying element conferring resistance to phenicols and oxazolidinones from Streptococcus parasuis, is transferable to Streptococcus suis

Streptococcus parasuis is a potential opportunistic zoonotic pathogen which is a close relative to Streptococcus suis, which exhibit extensive genetic exchange. The occurrence and dissemination of oxazolidinone resistance poses a severe threat to public health. However, such knowledge about the optrA gene in S. parasuis is limited. Herein, we characterized an optrA-positive multi-resistant S. parasuis isolate AH0906, in which the capsular polysaccharide locus exhibited a hybrid structure of S. suis serotype 11 and S. parasuis serotype 26. The optrA and erm(B) genes were co-located on a novel ICE of the ICESsuYZDH1 family, designated ICESpsuAH0906. IS1216E-optrA-carrying translocatable unit could be formed when excised from ICESpsuAH0906. ICESpsuAH0906 was found to be transferable from isolate AH0906 to Streptococcus suis P1/7RF at a relative high frequency of ∼ 10-5. Nonconservative integrations of ICESpsuAH0906 into the primary site SSU0877 and secondary site SSU1797 with 2-/4-nt imperfect direct repeats in recipient P1/7RF were observed. Upon transfer, the transconjugant displayed elevated MICs of the corresponding antimicrobial agents and performed a weak fitness cost when compared with the recipient strain. To our knowledge, it is the first description of the transfer of optrA in S. prarasuis and the first report of interspecies transfer of ICE with triplet serine integrases (of the ICESsuYZDH1 family). Considering the high transmission frequency of the ICEs and the extensive genetic exchange potential of S. parasuis with other streptococci, attention should be paid to the dissemination of the optrA gene from S. parasuis to clinically more important bacterial pathogens.

Genome-Wide Characterization of the R2R3-MYB Gene Family in Diospyros oleifera

The MYB gene family is one of the largest transcription factor families, which is clustered into four subfamilies according to the number of imperfect amino acid sequences repeats in their conserved MYB domain. R2R3-MYB is the largest subfamily that plays a diverse role in plant growth and development as well as adversity stresses. Diospyros has a wide range of applications in biomedical science and the food, wood, and chemical industries. Among these species, Diospyros oleifera can be used as a model plant for the Diospyros genus and the Ebenaceae family. Although the genome sequence of Diospyros oleifera was recently published in our previous work, bioinformatics and expression pattern analysis of the MYB gene family are limited. Here, we present the findings of a genome-wide analysis and the expression profiles of the R2R3-MYB transcription factor in Diospyros oleifera. A total of 129 R2R3-MYB genes were identified and classified into 28 groups (C1–C28) which had conserved motifs. The subfamily genes were unevenly distributed in 15 chromosomes; chromosome 6 and 7 have the most DoMYB genes. A total of 44 fragment replication events containing 57 DoMYB genes were identified using synteny analysis. In addition, collinear analysis revealed that 70 (54%) pairs of R2R3-MYB genes of Diospyros oleifera were collinear with Arabidopsis thaliana. Upon combining the data from RNA-seq and qRT-PCR, four key genes were screened and identified to correlate with the soluble tannin content during fruit development. DoMYB22 may be related to the synthesis of soluble tannin in persimmon. These results lay an important foundation for further studies on the R2R3-MYB gene function in persimmon fruit development.

Dissecting the Self-assembly Dynamics and Conformations of Each Imperfect Repeat of α-Synuclein

Dissecting the Self-assembly Dynamics and Conformations of Each Imperfect Repeat of α-Synuclein

Genome-Wide Identification and Expression Analysis of the R2R3-MYB Gene Family in Rubber Trees

The plant MYB transcription factor family featured as highly conserved DNA-binding domains consisting of 1 to 4 imperfect repeats (R). Increasing evidence indicates that MYB genes participates in growth, differentiation, metabolism, and biotic and abiotic stress responses. However, the functions of MYB genes in the rubber tree remain to be deeply elucidated, especially R2R3-MYB gene family. In this study, molecular biology, bioinformatics, and qRT-PCR were used to identify and analyze HbR2R3-MYB gene family members in the rubber tree. A total of 132 members of the R2R3-MYB gene family were identified in the rubber tree based on genome-wide level. Most of the HbR2R3-MYBs were mapped to 17 rubber tree chromosomes except four genes. A phylogenetic analysis divided all the HbR2R3-MYBs into 20 subgroups with Arabidopsis thaliana. MEME analysis showed that the protein of HbR2R3-MYBs was characterized by 9 conserved motifs. Twenty-six representative R2R3 HbMYBs from different subgroups were selected for expression profiles analysis and the results revealed that the HbR2R3-MYBs members showed various expression patterns in different tissues, powdery mildew-infected and ethylene treatment, implying the diversity of their functions in rubber trees. These results provide fundamental knowledge for further studying the response of the HbR2R3-MYB family to stress and regulation latex flow in rubber tree.

Insertion sequences related to ISAjo2 target pdif and dif sites and belong to a new IS family, the IS1202 family.

Several insertion sequences (IS) found in various Acinetobacter species exhibit target specificity. They are found, in the same orientation, 5 bp from the XerC binding site of the pdif sites associated with dif modules in Acinetobacter plasmids, and searches revealed they are also found near chromosomal dif sites of Acinetobacter species. These IS are 1.5 kb long, bounded by 24-26 bp imperfect terminal inverted repeats (TIRs) and encode a large transposase of 441-457 aa. They generate 5 bp target site duplications (TSDs). Structural predictions of the ISAjo2 transposase, TnpAjo2, modelled on TnsB of Tn7 revealed two N-terminal HTH domains followed by an RNaseH fold (DDE domain), a β barrel and a C-terminal domain. Similar to Tn7, the outer IS ends are 5'-TGT and ACA-3', and an additional Tnp binding site, corresponding to the internal portion of the IR, is found near each end. However, the Acinetobacter IS do not encode further proteins related to those required by Tn7 for targeted transposition, and the transposase may interact directly with XerC bound to a dif-like site. We propose that these IS, currently in the IS1202 group in the not characterized yet (NCY) category in ISFinder, are part of a distinct IS1202 family. Other IS listed as in the IS1202 group encode transposases related to TnpAjo2 (25-56 % amino acid identity) and have similar TIRs but fall into three groups based on the TSD length (3-5, >15, 0 bp). Those with 3-5 bp TSDs may also target dif-like sites but targets were not found for the other groups.

The Telomeric Repeats of HHV-6A Do Not Determine the Chromosome into Which the Virus Is Integrated

Human herpes virus 6A (HHV-6A) is able to integrate into the telomeric and subtelomeric regions of human chromosomes representing chromosomally integrated HHV-6A (ciHHV-6A). The integration starts from the right direct repeat (DRR) region. It has been shown experimentally that perfect telomeric repeats (pTMR) in the DRR region are required for the integration, while the absence of the imperfect telomeric repeats (impTMR) only slightly reduces the frequency of HHV-6 integration cases. The aim of this study was to determine whether telomeric repeats within DRR may define the chromosome into which the HHV-6A integrates. We analysed 66 HHV-6A genomes obtained from public databases. Insertion and deletion patterns of DRR regions were examined. We also compared TMR within the herpes virus DRR and human chromosome sequences retrieved from the Telomere-to-Telomere consortium. Our results show that telomeric repeats in DRR in circulating and ciHHV-6A have an affinity for all human chromosomes studied and thus do not define a chromosome for integration.

Electrostatic interactions mediate the nucleation and growth of a bacterial functional amyloid.

Bacterial biofilm formation can have severe impacts on human and environmental health. Enteric bacteria produce functional amyloid fibers called curli that aid in biofilm formation and host colonization. CsgA is the major proteinaceous component of curli amyloid fibers and is conserved in many gram-negative enteric bacteria. The CsgA amyloid core consists of five imperfect repeats (R1-R5). R2, R3, and R4 have aspartic acid (D) and glycine (G) residues that serve as "gatekeeper" residues by modulating the intrinsic aggregation propensity of CsgA. Here, using mutagenesis, salt-mediated charge screening, and by varying pH conditions, we show that the ability of CsgA variants to nucleate and form amyloid fibers is dictated by the charge state of the gatekeeper residues. We report that in Citrobacter youngae CsgA, certain arginine (R) and lysine (K) residues also act as gatekeeper residues. A mechanism of gatekeeping is proposed wherein R and K residues electrostatically interact with negatively charged D residues, tempering CsgA fiber formation.

Molecular Insights into a Novel Cu(I)-Sensitive ArsR/SmtB Family Repressor in Extremophile Acidithiobacillus caldus.

Acidithiobacillus caldus is a common bioleaching bacterium that is inevitably exposed to extreme copper stress in leachates. The ArsR/SmtB family of metalloregulatory repressors regulates homeostasis and resistance in bacteria by specifically responding to metals. Here, we characterized A. caldus Cu(I)-sensitive repressor (AcsR) and gained molecular insights into this new member of the ArsR/SmtB family. Transcriptional analysis indicated that the promoter (PIII) of acsR was highly active in Escherichia coli but inhibited upon AcsR binding to the PIII-acsR region. Size exclusion chromatography and circular dichroism spectra revealed that CuI-AcsR shared an identical assembly state with apo-AcsR, as a dimer with fewer α helices, more extended strands, and more β turns. Mutation of the cysteine site in AcsR did not affect its assembly state. Copper(I) titrations revealed that apo-AcsR bound two Cu(I) molecules per monomer in vitro with an average dissociation constant (KD) for bicinchoninic acid competition of 2.55 × 10-9 M. Site-directed mutation of putative Cu(I)-binding ligands in AcsR showed that replacing Cys64 with Ala reduces copper binding ability from two Cu(I) molecules per monomer to one, with an average KD of 6.05 × 10-9 M. Electrophoretic mobility shift assays revealed that apo-AcsR has high affinity for the 12-2-12 imperfect inverted repeats P2245 and P2270 in the acsR gene cluster and that Cu-loaded AcsR had lower affinity for DNA fragments than apo-AcsR. We developed a hypothetical working model of AcsR to better understand Cu resistance mechanisms in A. caldus. IMPORTANCE Copper (Cu) resistance among various microorganisms is attracting interest. The chemolithoautotrophic bacterium A. caldus, which can tolerate extreme copper stress (≥10 g/L Cu ions), is typically used to bioleach chalcopyrite (CuFeS2). Understanding of Cu resistance in A. caldus is limited due to scant investigation and the absence of efficient gene manipulation tools. Here, we characterized a new member of the ArsR/SmtB family of prokaryotic metalloregulatory transcriptional proteins that repress operons linked to stress-inducing concentrations of heavy metal ions. This protein can bind two Cu(I) molecules per monomer and negatively regulate its gene cluster. Members of the ArsR/SmtB family have not been investigated in A. caldus until now. The discovery of this novel protein enriches understanding of Cu homeostasis in A. caldus.

Time-lapse seismic imaging using shot gathers with nonrepeatable source wavelets

In time-lapse seismic applications, the signal produced by changes in the properties of subsurface rocks is generally obscured by noise associated with imperfect repeatability between surveys. A particularly important obstacle in the formation of time-lapse difference images is variation in the effective source wavelet between baseline and monitoring data sets. However, the partially separable influence of the wavelet within Green’s function model of seismic data permits two frequency-domain matching filters to be designed, which act to reduce source wavelet nonrepeatability. One is based on the spectral ratio of the baseline and monitoring wavelets and can be applied when prior estimates of the wavelets are available; the other is the average spectral ratio of the baseline and monitoring traces and can be applied when prior estimates are unavailable. After balancing the data sets with either of these filters, we further prepare for the imaging step with time-shift corrections, using a published fast local crosscorrelations algorithm, preparing the difference data for use in an imaging algorithm. Reverse time migration is engaged for the imaging task, but we observe that residual repeatability errors tend to be magnified at this stage when source-normalized crosscorrelation imaging conditions are used. Testing indicates that replacing this with a Poynting vector imaging condition strongly suppresses remaining errors in a robust manner. This includes stability within simulated data environments to noise-free data and data with random noise, up to signal-to-noise ratios of roughly one. Furthermore, our method illustrates better performance when compared with the conventional least-squares matching filter and common-depth-point-domain warping. At present, there is no common workflow for seismic imaging directly using time-lapse shot gathers. Our research contribution lies not only in the two matching filters but also in a novel workflow for time-lapse seismic imaging.

Shapeshifting tau: from intrinsically disordered to paired-helical filaments.

Tau is an intrinsically disordered protein that has the ability to self-assemble to form paired helical and straight filaments in Alzheimer's disease, as well as the ability to form additional distinct tau filaments in other tauopathies. In the presence of microtubules, tau forms an elongated form associated with tubulin dimers via a series of imperfect repeats known as the microtubule binding repeats. Tau has recently been identified to have the ability to phase separate in vitro and in cells. The ability of tau to adopt a wide variety of conformations appears fundamental both to its biological function and also its association with neurodegenerative diseases. The recently highlighted involvement of low-complexity domains in liquid-liquid phase separation provides a critical link between the soluble function and the insoluble dysfunctional properties of tau.

The mutational dynamics of short tandem repeats in large, multigenerational families

BackgroundShort tandem repeats (STRs) compose approximately 3% of the genome, and mutations at STR loci have been linked to dozens of human diseases including amyotrophic lateral sclerosis, Friedreich ataxia, Huntington disease, and fragile X syndrome. Improving our understanding of these mutations would increase our knowledge of the mutational dynamics of the genome and may uncover additional loci that contribute to disease. To estimate the genome-wide pattern of mutations at STR loci, we analyze blood-derived whole-genome sequencing data for 544 individuals from 29 three-generation CEPH pedigrees. These pedigrees contain both sets of grandparents, the parents, and an average of 9 grandchildren per family.ResultsWe use HipSTR to identify de novo STR mutations in the 2nd generation of these pedigrees and require transmission to the third generation for validation. Analyzing approximately 1.6 million STR loci, we estimate the empirical de novo STR mutation rate to be 5.24 × 10−5 mutations per locus per generation. Perfect repeats mutate about 2 × more often than imperfect repeats. De novo STRs are significantly enriched in Alu elements.ConclusionsApproximately 30% of new STR mutations occur within Alu elements, which compose only 11% of the genome, but only 10% are found in LINE-1 insertions, which compose 17% of the genome. Phasing these mutations to the parent of origin shows that parental transmission biases vary among families. We estimate the average number of de novo genome-wide STR mutations per individual to be approximately 85, which is similar to the average number of observed de novo single nucleotide variants.

New cellular T-DNAs in naturally transgenic plants

Naturally transgenic plants represent the result of Agrobacterium-mediated gene transfer. T-DNA of soil bacteria Agrobacterium integrated into plants genome is called cellular T-DNA (cT-DNA) [1]. Today, more than 50 species of naturally transgenic plants, or natural GMO (nGMO) are known [2, 3]. The function of cT-DNA in plants remains unknown. It is assumed that the fixation of transgenes could give plants different selective advantages depending on which genes had been integrated into the plant [4]. In order to clarify this issue, it is necessary to study more naturally transgenic plants. Until recently, the list of nGM plants contained less than 2 dozen species, but a search through genomic and transriptomic sequencing data made it possible to more than double this list [2]. In this work, we used the same approach, looking for cT-DNA genes in whole genome sequencing data that have appeared in the NCBI WGS since 2021. We found 14 new species of naturally transgenic plants, among which the most extended cT-DNAs were found in Triadica sebifera, Lonicera japonica, and Lonicera maackii. The cT-DNAs in these species are organized as imperfect inverted repeats. In the genomes of the species Paulownia fortunei, Apocynum venetum, Elaeagnus angustifolia, Erythroxylum havanense, E. densum, E. daphnites, E. cataractarum, Ceriops decandra, Camellia oleifera, Silene uniflora, short cT-DNAs containing only opine synthesis genes were found. We also estimated the approximate age of the cT-DNAs. The first described examples date back to the Late Paleogene, and the process continues to the present. Thus, we can conclude that natural GMOs are a widespread phenomenon, many aspects of which remain unclear, requiring additional research on the topic.
 The article was made with support of the Ministry of Science and Higher Education of the Russian Federation in accordance with agreement No. 075-15-2022-322 dated 22.04.2022 on providing a grant in the form of subsidies from the federal budget of the Russian Federation. The grant was provided for state support for the creation and development of a world-class scientific center, Agrotechnologies for the Future.

Identifying the Gene Regulatory Network of the Starvation-Induced Transcriptional Activator Nla28.

Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a nla28 mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of in vitro promoter binding assays, coupled with in vitro and in vivo mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed in vivo targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. IMPORTANCE In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus. We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type.