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Despite the significant presence of plant-derived tricarboxylic acids in some environments, few studies detail the bacterial metabolism of trans-aconitic acid (Taa) and tricarballylic acid (Tcb). In a soil bacterium, Acinetobacter baylyi ADP1, we discovered interrelated pathways for the consumption of Taa and Tcb. An intricate regulatory scheme tightly controls the transport and catabolism of both compounds and may reflect that they can be toxic inhibitors of the tricarboxylic acid cycle. The genes encoding two similar LysR-type transcriptional regulators, TcuR and TclR, were clustered on the chromosome with tcuA and tcuB, genes required for Tcb consumption. The genetic organization differed from that in Salmonella enterica serovar Typhimurium, in which tcuA and tcuB form an operon with a transporter gene, tcuC. In A. baylyi, tcuC was not cotranscribed with tcuAB. Rather, tcuC was cotranscribed with a gene, designated pacI, encoding an isomerase needed for Taa consumption. TcuC appears to transport Tcb and cis-aconitic acid (Caa), the presumed product of PacI-mediated periplasmic isomerization of Taa. Two operons, tcuC-pacI and tcuAB, were transcriptionally controlled by both TcuR and TclR, which have overlapping functions. We investigated the roles of the two regulators in activating transcription of both operons in response to multiple effector compounds, including Taa, Tcb, and Caa. [ABSTRACT FROM AUTHOR]

l-cysteine biosynthesis from inorganic sulfur represents a major mechanism by which reduced sulfur is incorporated into organic compounds. Cysteine biosynthesis and regulation is well characterized in Escherichia coli. However, the regulation of sulfur metabolism in Acinetobacter baumannii is only partly understood, with the LysR-type regulator, GigC known to control some aspects of sulfur reduction. In this study, we have used transcriptomics and bioinformatic analyses to characterize a novel LysR-type transcriptional regulator encoded by ABUW_1016 (cbl), in a highly multidrug resistant and virulent isolate of A. baumannii. We have shown that Cbl is involved in controlling expression of the genes required for uptake and reduction of various sulfur sources in A. baumannii. Collectively, we have identified the global regulon of Cbl and proposed a model of cysteine biosynthesis and its regulation by Cbl and GigC in A. baumannii. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacteria have evolved a sophisticated array of signal transduction systems that allow them to adapt their physiology and metabolism to changing environmental conditions. Typically, these systems recognize signals through dedicated ligand binding domains (LBDs) to ultimately trigger a diversity of physiological responses. Nonetheless, an increasing number of reports reveal that signal transduction receptors also bind antagonists to inhibit responses mediated by agonists. The mechanisms by which antagonists block the downstream signaling cascade remain largely unknown. To advance our knowledge in this field, we used the LysR-type transcriptional regulator AdmX as a model. AdmX activates the expression of an antibiotic biosynthetic cluster in the rhizobacterium Serratia plymuthica. AdmX specifically recognizes the auxin phytohormone indole-3-acetic acid (IAA) and its biosynthetic intermediate indole-3-pyruvic acid (IPA) as signals. However, only IAA, but not IPA, was shown to regulate antibiotic production in S. plymuthica. Here, we report the high-resolution structures of the LBD of AdmX in complex with IAA and IPA. We found that IAA and IPA compete for binding to AdmX. Although IAA and IPA binding does not alter the oligomeric state of AdmX, IPA binding causes a higher degree of compactness in the protein structure. Molecular dynamics simulations revealed significant differences in the binding modes of IAA and IPA by AdmX, and the inspection of the three-dimensional structures evidenced differential agonist- and antagonist-mediated structural changes. Key residues for auxin binding were identified and an auxin recognition motif defined. Phylogenetic clustering supports the recent evolutionary emergence of this motif specifically in plant-associated enterobacteria. IMPORTANCE Although antagonists were found to bind different bacterial signal transduction receptors, we are still at the early stages of understanding the molecular details by which these molecules exert their inhibitory effects. Here, we provide insight into the structural changes resulting from the binding of an agonist and an antagonist to a sensor protein. Our data indicate that agonist and antagonist recognition is characterized by small conformational differences in the LBDs that can be efficiently transmitted to the output domain to modulate the final response. LBDs are subject to strong selective pressures and are rapidly evolving domains. An increasing number of reports support the idea that environmental factors drive the evolution of sensor domains. Given the recent evolutionary history of AdmX homologs, as well as their narrow phyletic distribution within plant-associated bacteria, our results are in accordance with a plant-mediated evolutionary process that resulted in the emergence of receptor proteins that specifically sense auxin phytohormones.

The regulated uptake and consumption of D-amino acids by bacteria remain largely unexplored, despite the physiological importance of these compounds. Unlike other characterized bacteria, such as Escherichia coli, which utilizes only L-Asp, Acinetobacter baylyi ADP1 can consume both D-Asp and L-Asp as the sole carbon or nitrogen source. As described here, two LysR-type transcriptional regulators (LTTRs), DarR and AalR, control D- and L-Asp metabolism in strain ADP1. Heterologous expression of A. baylyi proteins enabled E. coli to use D-Asp as the carbon source when either of two transporters (AspT or AspY) and a racemase (RacD) were coexpressed. A third transporter, designated AspS, was also discovered to transport Asp in ADP1. DarR and/or AalR controlled the transcription of aspT, aspY, racD, and aspA (which encodes aspartate ammonia lyase). Conserved residues in the N-terminal DNA-binding domains of both regulators likely enable them to recognize the same DNA consensus sequence (ATGC-N7-GCAT) in several operator-promoter regions. In strains lacking AalR, suppressor mutations revealed a role for the ClpAP protease in Asp metabolism. In the absence of the ClpA component of this protease, DarR can compensate for the loss of AalR. ADP1 consumed L- and D-Asn and L-Glu, but not D-Glu, as the sole carbon or nitrogen source using interrelated pathways. [ABSTRACT FROM AUTHOR]

Abstract Small molecules are able to regulate numerous cellular processes through binding to various bacterial receptor proteins, but the mechanisms and functions by which these chemicals coordinate and execute remain poorly understood. 4-hydroxybenzoic acid (4-HBA) and cyclic di-GMP (c-di-GMP) are two such molecules with distinct structures that are produced in Lysobacter enzymogenes to synergistically affect the secretion of an antifungal antibiotic, known as heat-stable antifungal factor (HSAF). In our earlier studies, we showed that CdgL, a YajQ-like protein without DNA-binding domain, was able to physically interact with LysR, a transcription factor, to enhance its binding affinity toward the upstream region of the HSAF biosynthesis operon promoter, hence increasing the HSAF biosynthesis. Interestingly, 4-HBA or c-di-GMP can bind to its cognate receptor of LysR or CdgL, respectively, to regulate the HSAF biosynthesis. Further, c-di-GMP acts by binding to CdgL to induce the dissociation of the CdgL-LysR complex, leading to decreased downstream expression. We now showed that CdgL controlled the transcription of lenB2, which encodes an oxygenase to convert chorismate to 4-HBA. Notably, overexpression of cdgL was found to stimulate lenB2 transcription, which likely increased the intracellular 4-HBA content. Also, 4-HBA could bind to LysR to interrupt the LysR-CdgL complex formation and release of CdgL, which caused a lower affinity of LysR toward DNA and hence decreased HSAF operon expression. These findings, along with our earlier report, allow us to propose a coordination mechanism demonstrating how the HSAF biosynthesis is co-regulated by 4-HBA and c-di-GMP through interactions with their cognate receptors. This new mechanism shall shed light on improving the HSAF yield for practical usage.

Summary: YajQ, a binding protein of the universal bacterial second messenger cyclic di‐GMP (c‐di‐GMP), affects virulence in several bacterial pathogens, including Xanthomonas campestris. In this bacterium, YajQ interacts with the transcription factor LysR. Upon c‐di‐GMP binding, the whole c‐di‐GMP‐YajQ‐LysR complex is found to dissociate from DNA, resulting in virulence gene regulation. Here, we identify a YajQ‐LysR‐like system in the bacterial biocontrol agent Lysobacter enzymogenes OH11 that secretes an antifungal antibiotic, heat‐stable antifungal factor (HSAF) against crop fungal pathogens. We show that the YajQ homologue, CdgL (c‐di‐GMP receptor interacting with LysR) affects expression of the HSAF biosynthesis operon by interacting with the transcription activator LysR. The CdgL‐LysR interaction enhances the apparent affinity of LysR to the promoter region upstream of the HSAF biosynthesis operon, which increases operon expression. Unlike the homologues CdgL (YajQ)‐LysR system in X. campestris, we show that c‐di‐GMP binding to CdgL seems to weaken CdgL‐LysR interactions and promote the release of CdgL from the LysR‐DNA complex, which leads to decreased expression. Together, this study takes the YajQ‐LysR‐like system from bacterial pathogens to a crop‐protecting bacterium that is able to regulate antifungal HSAF biosynthesis via disassembly of the c‐di‐GMP receptor–transcription activator complex. [ABSTRACT FROM AUTHOR]

A simple aspirin-inducible system has been developed and characterised in E. coli by employing the Psal promoter and SalR regulation system originally from Acinetobacter baylyi ADP1. Mutagenesis at the DNA binding domain (DBD) and chemical recognition domain (CRD) of the SalR protein in A. baylyi ADP1 suggests that the effector free form SalRr could compete with the effector bound form SalRa, binding the Psal promoter and repress gene transcription. The induction of the Psal promoter was compared in two different gene-circuit designs: simple regulation system (SRS) and positive autoregulation (PAR). Both regulatory circuits were induced in a dose-dependent manner in the presence of aspirin in the range of 0.05-10 µM. Over-expression of SalR in the SRS circuit reduced both baseline leakiness and the strength of Psal promoter. The PAR circuit forms a positive feedback loop that fine-tunes the level of SalR. A mathematical simulation based on SalRr/SalRa competitive binding model not only fit the observed experimental results in SRS and PAR circuits, but also predicted the performance of a new gene circuit design, for which weak expression of SalR in the SRS circuit should significantly improve the induction strength. The experimental result is in a good agreement with this prediction, validating the SalRr/SalRa competitive binding model. The aspirin-inducible systems were also functional in probiotic strain E.coli Nissle 1917 (EcN) and SimCells produced from E. coli MC1000 ΔminD. These well-characterised and modularised aspirin-inducible gene circuits would be useful biobricks to synthetic bology. [ABSTRACT FROM AUTHOR]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) catalyzes the first major step of carbon fixation in the Calvin-Benson-Bassham (CBB) cycle. This autotrophic CO2 fixation cycle accounts for almost all the assimilated carbon on Earth. Due to the primary role that RubisCO plays in autotrophic carbon fixation, it is important to understand how its gene expression is regulated and the enzyme is activated. Since the majority of all microorganisms are currently not culturable, we used a metagenomic approach to identify genes and enzymes associated with RubisCO expression. The investigated metagenomic DNA fragment originates from the deep-sea hydrothermal vent field Nibelungen at 8°18′ S along the Mid-Atlantic Ridge. It is 13,046 bp and resembles genes from Thiomicrospira crunogena. The fragment encodes nine open reading frames (ORFs) which include two types of RubisCO, form I (CbbL/S) and form II (CbbM), two LysR transcriptional regulators (LysR1 and LysR2), two von Willebrand factor type A (CbbO-m and CbbO-1), and two AAA+ ATPases (CbbQ-m and CbbQ-1), expected to function as RubisCO activating enzymes. In silico analyses uncovered several putative LysR binding sites and promoter structures. Functions of some of these DNA motifs were experimentally confirmed. For example, according to mobility shift assays LysR1’s binding ability to the intergenic region of lysR1 and cbbL appears to be intensified when CbbL or LysR2 are present. Binding of LysR2 upstream of cbbM appears to be intensified if CbbM is present. Our study suggests that CbbQ-m and CbbO-m activate CbbL and that LysR1 and LysR2 proteins promote CbbQ-m/CbbO-m expression. CbbO-1 seems to activate CbbM and CbbM itself appears to contribute to intensifying LysR’s binding ability and thus its own transcriptional regulation. CbbM furthermore appears to impair cbbL expression. A model summarizes the findings and predicts putative interactions of the different proteins influencing RubisCO gene regulation and expression.

Background: 3-Hydroxypropionic acid (3-HP) is an important platform chemical that boasts a variety of industrial applications. Gene expression systems inducible by 3-HP, if available, are of great utility for optimization of the pathways of 3-HP production and excretion. Results: Here we report the presence of unique inducible gene expression systems in Pseudomonas denitrificans and other microorganisms. In P. denitrificans, transcription of three genes (hpdH, mmsA and hbdH-4) involved in 3-HP degradation was upregulated by 3-HP by the action of a transcriptional regulator protein, LysR, and a cis-acting regulatory site for LysR binding. Similar inducible systems having an LysR transcriptional regulator were identified in other microorganisms that also could degrade 3-HP. A docking study showed that the 3-HP binding pocket is located between the N-terminal helix-turn-helix motif and the C-terminal cofactor-binding domain. Conclusions: This LysR-regulated 3-HP-inducible system should prove useful for control of the level of gene expression in response to 3-HP. [ABSTRACT FROM AUTHOR]

The protein family of LysR-type transcriptional regulators (LTTRs) is highly abundant among prokaryotes. We analyzed 10,145 non-redundant microbial sequences with homology to eight LysR family regulators of a model prokaryote, Geobacter sulfurreducens, and employed phylogenetic tree inference for LTTR classification. We also analyzed the arrangement of genome clusters containing G. sulfurreducens LTTR genes and searched for LTTR regulatory motifs, suggesting likely regulatory targets of G. sulfurreducens LTTRs . This is the first study to date providing a detailed classification of LTTRs in the deltaproteobacterial family Geobacteraceae. [ABSTRACT FROM AUTHOR]

Summary: In Klebsiella pneumoniae M5a1, mhbTDHIM genes are involved in 3-hydroxybenzoate catabolism via the gentisate pathway. mhbR, which encodes a LysR-type transcriptional regulator, is divergently transcribed from the mhb structural genes. MhbR was found to be necessary for the expression of catabolic genes. Transcriptional studies demonstrated that the mhb structural genes are transcribed as an operon. The promoters of mhbR and the mhb operon are σ 70-type and overlap with each other. 5′ Deletion analysis of the promoter transcription activity showed that a 233bp fragment (position −144 to +89 according to the transcriptional start site of mhb operon) contained the element necessary for induction. β-Galactosidase activity assays and electrophoretic mobility shift assays showed that an inverted repeat sequence site 1 (ATAACCTCCAGGTTAT, position −70 to −55) within this fragment was critical for regulation. [Copyright &y& Elsevier]

Abstract: Cells have devised a variety of protection systems against the toxic effects of dioxygen. Dioxygenases are part of this defence mechanism. In Escherichia coli, the positive regulator HcaR, a member of the LysR family of regulators, controls expression of the neighbouring genes, hcaA1, hcaA2, hcaC, hcaB and hcaD, coding for the 3-phenylpropionate dioxygenase complex and 3-phenylpropionate-2′,3′-dihydrodiol dehydrogenase, that oxidizes 3-phenylpropionate to 3-(2,3-dihydroxyphenyl) propionate. Differences between expression of hcaR and expression of its target, hcaA, suggest that HcaR is involved in control of other cellular processes or that other regulatory proteins modulate hcaA expression. Protein expression profiling was used to identify other HcaR targets. Two-dimensional gel electrophoresis was used to compare the proteomes of wild-type E. coli and strains in which hcaR was disrupted. Several polypeptides whose production was up- or downregulated in the hcaR mutant were involved in the oxidative stress response. Subsequent experiments demonstrated that hcaR disruption was involved in regulation of genes involved in the oxidative stress response. Modification of the stress response also occurred in an hcaA1A2CD mutant strain. Using gel retardation, the HcaR binding site was estimated to be located about −70 to −55 bp upstream of the hcaA transcription start site. The expression of hcaR was repressed in the absence of oxygen by the ArcA/ArcB two-component system. [Copyright &y& Elsevier]

The invasion and colonization of host plants by the destructive pathogen Ralstonia solanacearum rely on its cell motility, which is controlled by multiple factors. Here, we report that the LysR-type transcriptional regulator CrgA (RS_RS16695) represses cell motility in R. solanacearum GMI1000. CrgA possesses common features of a LysR-type transcriptional regulator and contains an N-terminal helix-turn-helix motif as well as a C-terminal LysR substrate-binding domain. Deletion of crgA results in an enhanced swim ring and increased transcription of flhDC In addition, the Δ crgA mutant possesses more polar flagella than wild-type GMI1000 and exhibits higher expression of the flagellin gene fliC Despite these alterations, the Δ crgA mutant did not have a detectable growth defect in culture. Yeast one-hybrid and electrophoretic mobility shift assays revealed that CrgA interacts directly with the flhDC promoter. Expressing the β-glucuronidase (GUS) reporter under the control of the crgA promoter showed that crgA transcription is dependent on cell density. Soil-soaking inoculation with the crgA mutant caused wilt symptoms on tomato ( Solanum lycopersicum L. cv. Hong yangli) plants earlier than inoculation with the wild-type GMI1000 but resulted in lower disease severity. We conclude that the R. solanacearum regulator CrgA represses flhDC expression and consequently affects the expression of fliC to modulate cell motility, thereby conditioning disease development in host plants. IMPORTANCE Ralstonia solanacearum is a widely distributed soilborne plant pathogen that causes bacterial wilt disease on diverse plant species. Motility is a critical virulence attribute of R. solanacearum , motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum , motility-defective strains are markedly affected in pathogenicity, which is coregulated with multiple virulence factors. In this study, we identified a new LysR-type transcriptional regulator (LTTR), CrgA, that negatively regulates motility. The mutation of the corresponding gene leads to the precocious appearance of wilt symptoms on tomato plants when the pathogen is introduced using soil-soaking inoculation. This study indicates that the regulation of R. solanacearum motility is more complex than previously thought and enhances our understanding of flagellum regulation in R. solanacearum ., (Copyright © 2020 American Society for Microbiology.)

The simultaneous response of one transcriptional regulator to different effectors remains largely unexplored. Nevertheless, such interactions can substantially impact gene expression by rapidly integrating cellular signals and by expanding the range of transcriptional responses. In this study, similarities between paralogs were exploited to engineer novel responses in CatM, a regulator that controls benzoate degradation in Acinetobacter baylyi ADP1. One goal was to improve understanding of how its paralog, BenM, activates transcription in response to two compounds (cis,cis-muconate and benzoate) at levels significantly greater than with either alone. Despite the overlapping functions of BenM and CatM, which regulate many of the same ben and cat genes, CatM normally responds only to cis,cis-muconate. Using domain swapping and site-directed amino acid replacements, CatM variants were generated and assessed for the ability to activate transcription. To create a variant that responds synergistically to both effectors required alteration of both the effector-binding region and the DNA-binding domain. These studies help define the interconnected roles of protein domains and extend understanding of LysR-type proteins, the largest family of transcriptional regulators in bacteria. Additionally, renewed interest in the modular functionality of transcription factors stems from their potential use as biosensors. [ABSTRACT FROM AUTHOR]

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc- gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR , a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc -gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.