Two LysR family transcriptional regulators, McbH and McbN, activate the operons responsible for the midstream and downstream pathways of carbaryl degradation in Pseudomonas sp. strain XWY-1, respectively

Previously, a LysR family transcriptional regulator McbG that activates the mcbBCDEF gene cluster involved in the upstream pathway (from carbaryl to salicylate) of carbaryl degradation in Pseudomonas sp. strain XWY-1 has been identified by us ( Appl. Environ. Microbiol. 2021, 87(9): e02970-20.). In this study, we identified McbH and McbN, which activate mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and mcbOPQ cluster (responsible for the downstream pathway, from gentisate to pyruvate and fumarate), respectively. They both belong to the LysR family of transcriptional regulators. Gene disruption and complementation study reveal that McbH is essential for transcription of the mcbIJKLM cluster in response to salicylate and McbN is indispensable for the transcription of the mcbOPQ cluster in response to gentisate. The results of electrophoretic mobility shift assay (EMSA) and DNase I footprinting showed that McbH binds to the 52-bp motif in the mcbIJKLM promoter area and McbN binds to the 58-bp motif in the mcbOPQ promoter area. The key sequence of McbH binding to mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of LysR family. However, the 12-bp motif that is different from the typical characteristics of the LysR family regulator binding site sequence is identified as the key sequence for McbN to bind to the mcbOPQ promoter. This study reveals the regulatory mechanism for the midstream and downstream pathway of carbaryl degradation in strain XWY-1 and further enriches the members of the LysR transcription regulator family. IMPORTANCE: The enzyme-encoding genes involved in the complete degradation pathway of carbaryl in Pseudomonas sp. strain XWY-1 include mcbABCDEF , mcbIJKLM and mcbOPQ . Previous studies demonstrated that the mcbA gene responsible for hydrolysis of carbaryl to 1-naphthol is constitutively expressed and the transcription of mcbBCDEF was regulated by McbG. However, the transcription regulation mechanisms of mcbIJKLM and mcbOPQ have not been investigated yet. In this study, we identified two LysR-type transcriptional regulators, McbH and McbN, which activate the mcbIJKLM cluster responsible for the degradation of salicylate to gentisate and mcbOPQ cluster responsible for the degradation of gentisate to pyruvate and fumarate, respectively. The 13-bp motif is critical for McbH to bind to the promoter of mcbIJKLM , and 12-bp motif different from the typical characteristics of the LTTR binding sequence affects the binding of McbN to promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.

Identification of a transcription factor, PunR, that regulates the purine and purine nucleoside transporter punC in E. coli

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, the analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. The inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine, inosine, or guanosine as the nitrogen source. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

A LysR family transcriptional regulator modulates biofilm formation and protease production in Burkholderia cenocepacia

Quorum sensing (QS) signals are widely employed by bacteria to regulate biological functions in response to cell densities. Previous studies showed that Burkholderia cenocepacia mostly utilizes two types of QS systems, including the N-acylhomoserine lactone (AHL) and cis-2-dodecenoic acid (BDSF) systems, to regulate biological functions. We demonstrated here that a LysR family transcriptional regulator Bcal3178 controls the QS-regulated phenotypes, including biofilm formation and protease production, in B. cenocepacia H111. Expression of Bcal3178 at transcriptional level was obviously down-regulated in both the AHL-deficient and BDSF-deficient mutant strains comparing to the wild-type H111 strain. It was further identified that Bcal3178 regulated target gene expression by directly binding to the promoter DNA regions. We also revealed that Bcal3178 was directly controlled by the AHL system regulator CepR. These results show that Bcal3178 is a new downstream component of the QS signaling network that modulates a subset of genes and functions co-regulated by the AHL and BDSF QS systems in B. cenocepacia. IMPORTANCE Burkholderia cenocepacia is an important opportunistic pathogen in humans, which utilizes the BDSF and AHL quorum sensing (QS) systems to regulate biological functions and virulence. We demonstrated here that a new downstream regulator Bcal3178 of the QS signaling network controls biofilm formation and protease production. Bcal3178 is a LysR family transcriptional regulator modulated by both the BDSF and AHL QS systems. Furthermore, Bcal3178 controls many target genes which are regulated by the QS systems in B. cenocepacia. Collectively, our findings depict a novel molecular mechanism with which QS systems regulate some target gene expression and biological functions by modulating the expression level of a LysR family transcriptional regulator in B. cenocepacia.

A novel transcription factor PunR and Nac are involved in purine and purine nucleoside transporter punC regulation in E. coli

Many genes in bacterial genomes are of unknown function, often referred to as y-genes. Recently, novel analytic methods have divided bacterial transcriptomes into independently modulated sets of genes (iModulons). Functionally annotated iModulons that contain y-genes lead to testable hypotheses to elucidate y-gene function. Inversely correlated expression of a putative transporter gene, ydhC, relative to purine biosynthetic genes, has led to the hypothesis that it encodes a purine-related transporter and revealed a LysR-family regulator, YdhB, with a predicted 23-bp palindromic binding motif. RNA-Seq analysis of a ydhB knockout mutant confirmed the YdhB-dependent activation of ydhC in the presence of adenosine. The deletion of either the ydhC or the ydhB gene led to a substantially decreased growth rate for E. coli in minimal medium with adenosine as the nitrogen source, as well as with inosine or guanosine. Taken together, we provide clear evidence that YdhB activates the expression of the ydhC gene that encodes a novel purine transporter in E. coli. We propose that the genes ydhB and ydhC be re-named as punR and punC, respectively.

McbG, a LysR Family Transcriptional Regulator, Activates the mcbBCDEF Gene Cluster Involved in the Upstream Pathway of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1

ABSTRACT Although enzyme-encoding genes involved in the degradation of carbaryl have been reported in Pseudomonas sp. strain XWY-1, no regulator has been identified yet. In the mcbABCDEF cluster responsible for the upstream pathway of carbaryl degradation (from carbaryl to salicylate), the mcbA gene is constitutively expressed, while mcbBCDEF is induced by 1-naphthol, the hydrolysis product of carbaryl by McbA. In this study, we identified McbG, a transcriptional activator of the mcbBCDEF cluster. McbG is a 315-amino-acid protein with a molecular mass of 35.7 kDa. It belongs to the LysR family of transcriptional regulators and shows 28.48% identity to the pentachlorophenol (PCP) degradation transcriptional activation protein PcpR from Sphingobium chlorophenolicum ATCC 39723. Gene disruption and complementation studies reveal that mcbG is essential for transcription of the mcbBCDEF cluster in response to 1-naphthol in strain XWY-1. The results of the electrophoretic mobility shift assay (EMSA) and DNase I footprinting show that McbG binds to the 25-bp motif in the mcbBCDEF promoter area. The palindromic sequence TATCGATA within the motif is essential for McbG binding. The binding site is located between the –10 box and the transcription start site. In addition, McbG can repress its own transcription. The EMSA results show that a 25-bp motif in the mcbG promoter area plays an important role in McbG binding to the promoter of mcbG. This study reveals the regulatory mechanism for the upstream pathway of carbaryl degradation in strain XWY-1. The identification of McbG increases the variety of regulatory models within the LysR family of transcriptional regulators. IMPORTANCE Pseudomonas sp. strain XWY-1 is a carbaryl-degrading strain that utilizes carbaryl as the sole carbon and energy source for growth. The functional genes involved in the degradation of carbaryl have already been reported. However, the regulatory mechanism has not been investigated yet. Previous studies demonstrated that the mcbA gene, responsible for hydrolysis of carbaryl to 1-naphthol, is constitutively expressed in strain XWY-1. In this study, we identified a LysR-type transcriptional regulator, McbG, which activates the mcbBCDEF gene cluster responsible for the degradation of 1-naphthol to salicylate and represses its own transcription. The DNA binding site of McbG in the mcbBCDEF promoter area contains a palindromic sequence, which affects the binding of McbG to DNA. These findings enhance our understanding of the mechanism of microbial degradation of carbaryl.

LysR Family regulator LttR Controls Conjugated Linoleic Acid Production by Directly Activating the cla Operon in Lactobacillus plantarum

Conjugated linoleic acids (CLA) have attracted more attention as functional lipids due to their potential physiological activities including anti-cancer, anti-inflammatory, anti-cardiovascular disease and anti-diabetes. Microbiological synthesis of CLA has become a compelling method due to its high isomer selectivity and convenient separation and purification process. In Lactobacillus plantarum, the generation of CLA from linoleic acids (LA) requires the combination of CLA oleate hydratase (CLA-HY), CLA short-chain dehydrogenase (CLA-DH) and CLA acetoacetate decarboxylase (CLA-DC) which are separately encoded by cla-hy, cla-dh and cla-dc. However, the regulatory mechanisms of CLA synthesis remain unknown. In this study, we found that a lysR-family transcriptional regulator LTTR directly bound to the promoter region of cla operon and activated the transcription of cla-dh and cla-dc. The binding motif was also predicted by bioinformatics analysis and verified by EMSA and DNase I footprinting assay. The lttR overexpression strain showed a 5-fold increase in CLA production. Moreover, we uncovered that the transcription of lttR is activated by LA. These results indicate that LttR senses LA and promotes CLA production by activating the transcription of cla-dh and cla-dc. This study reveals a new regulatory mechanism in CLA biotransformation and provides a new potential metabolic engineering strategy to increase the yield of CLA. Importance Our work has identified a novel transcriptional regulator LTTR that regulates the production of CLA by activating the transcription of cla-dh and cla-dc, essential genes participating in the CLA synthesis in Lactobacillus plantarum. This provides the insight into the regulatory mechanism of CLA synthesis and broadens our understanding about synthesis and regulatory mechanisms of biosynthesis of CLA.

A Novel LysR Family Factor STM0859 is Associated with The Responses of Salmonella Typhimurium to Environmental Stress and Biofilm Formation

Salmonella enterica subsp. enterica serovar Typhimurium (ST) is an intracellularly parasitic bacterium. This zoonotic pathogen causes food poisoning and thus imposes a severe threat to food safety. Here, to understand the regulatory roles of the novel transcription factor STM0859 on the response of ST to environmental stress and biofilm formation, the STM0859 gene-deficient strain and the complementation strain ΔSTM0859/STM0859 were generated, respectively. Then, its capacity of responding to environmental stresses and biofilm (BF) formation ability under different stresses, including acid, alkali, high salt, cholate, and oxidative stresses was tested. We further analyzed the interaction between the STM0859 protein and the promoter of the acid stress response-related gene rcsB by performing an electrophoresis mobility shift assay (EMSA). The results showed that acid resistance and BF formation capacities of ST-ΔSTM0859 strain were significantly weaker, as compared with those of Salmonella Typhimurium SL1344 (ST-SL1344) wild strain (p < 0.01). Quantitative qRT-PCR analysis showed that the expression levels of acid stress and BF formation-related genes, rcsB and rpoS, of ST-ΔSTM0859 strain were significantly reduced at the transcription levels, while the transcription levels of these genes were fully restored in complementation strain ST-ΔSTM0859/STM0859. The results of EMSA showed that STM0859 was capable of binding the promoter DNA fragments of the rcsB gene, suggesting that STM0859 can promote the transcription of the rcsB gene through interaction with its promoter, thereby exerting an indirectly regulatory role on the adaptive responses to acid stress and BF formation of ST. This study provided new insights into the regulatory mechanisms of the LysR family factors on the tolerances of ST under adverse environmental stresses.

GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii

ABSTRACTA critical facet of mammalian innate immunity involves the hosts’ attempts to sequester and/or limit the availability of key metabolic products from pathogens. For example, nutritional immunity encompasses host approaches to limit the availability of key heavy metal ions such as zinc and iron. Previously, we identified several hundred genes in a multidrug-resistant isolate of Acinetobacter baumannii that are required for growth and/or survival in the Galleria mellonella infection model. In the present study, we further characterize one of these genes, a LysR family transcription regulator that we previously named GigC. We show that mutant strains lacking gigC have impaired growth in the absence of the amino acid cysteine and that gigC regulates the expression of several genes involved in the sulfur assimilation and cysteine biosynthetic pathways. We further show that cells harboring a deletion of the gigC gene are attenuated in two murine infection models, suggesting that the GigC protein, likely through its regulation of the cysteine biosynthetic pathway, plays a key role in the virulence of A. baumannii.

The Genome Sequence of Brucella abortus Vaccine Strain A19 Provides Insights on its Virulence Attenuation Compared to Brucella Abortus Strain 9-941

Background: Brucellosis is a widespread disease that affects animals and humans. The live attenuated Brucella abortus A19 strain is used for vaccination against brucellosis in China. In addition, the main mechanisms supporting the residual toxicity of A19 have not been elucidated. Here, we performed a comprehensive comparative analysis of the genome-wide sequence of A19 against the whole genome sequences of the published virulent reference strain 9-941. The primary objective of this study was to identify candidate virulence genes by systematically comparing the genomic sequences between the two genomes. Results: This analysis revealed two deletion regions in the A19 genome, in which all included large fragments of 63 bp, and one of their gene function is related to ABC transporter permease protein. In addition, we have identified minor mutations in important virulence-related genes that can be used to determine the underlying mechanisms of virulence attenuation. The function of its virulence gene covers LysR family transcriptional regulator, outer membrane, MFS transporter and oxidoreductase etc. At the same time, a PCR differential diagnosis method was constructed, which can distinguish A19, S19 and most other commonly used Brucella viruent strains and vaccine strains. Conclusion: The data may help to provide resources for further detailed analysis of mechanisms for other Brucella vaccines. It laid the foundation for further distinguishing between vaccine immunity and virulent strains infection.