LysR Family Of Transcriptional Regulators Research Articles

A Sb(III)-specific efflux transporter from Ensifer adhaerens E-60

Antimony is pervasive environmental toxic substance, and numerous genes encoding mechanisms to resist, transform and extrude the toxic metalloid antimony have been discovered in various microorganisms. Here we identified a major facilitator superfamily (MFS) transporter, AntB, on the chromosome of the arsenite-oxidizing bacterium Ensifer adhaerens E-60 that confers resistance to Sb(III) and Sb(V). The antB gene is adjacent to gene encoding a LysR family transcriptional regulator termed LysRars, which is an As(III)/Sb(III)-responsive transcriptional repressor that is predicted to control expression of antB. Similar antB and lysRars genes are found in related arsenic-resistant bacteria, especially strains of Ensifer adhaerens, and the lysRars gene adjacent to antB encodes a member of a divergent subgroup of putative LysR-type regulators. Closely related AntB and LysRars orthologs contain three conserved cysteine residues, which are Cys17, Cys99, and Cys350 in AntB and Cys81, Cys289 and Cys294 in LysRars, respectively. Expression of antB is induced by As(III), Sb(III), Sb(V) and Rox(III) (4-hydroxy-3-nitrophenyl arsenite). Heterologous expression of antB in E. coli AW3110 (Δars) conferred resistance to Sb(III) and Sb(V) and reduced the intracellular concentration of Sb(III). The discovery of the Sb(III) efflux transporter AntB enriches our knowledge of the role of the efflux transporter in the antimony biogeochemical cycle.

The LysR family transcriptional regulator ORF-L16 regulates spinosad biosynthesis in Saccharopolyspora spinosa

Spinosad, a potent broad-spectrum bioinsecticide produced by Saccharopolyspora spinosa, has significant market potential. Despite its effectiveness, the regulatory mechanisms of spinosad biosynthesis remain unclear. Our investigation identified the crucial role of the LysR family transcriptional regulator ORF-L16, located upstream of spinosad biosynthetic genes, in spinosad biosynthesis. Through reverse transcription PCR (RT-PCR) and 5′-rapid amplification of cDNA ends (5′-Race), we unveiled that the spinosad biosynthetic gene cluster (BGC) contains six transcription units and seven promoters. Electrophoretic mobility shift assays (EMSAs) demonstrated that ORF-L16 bound to seven promoters within the spinosad BGC, indicating its involvement in regulating spinosad biosynthesis. Notably, deletion of ORF-L16 led to a drastic reduction in spinosad production from 1818.73 mg/L to 1.69 mg/L, accompanied by decreased transcription levels of spinosad biosynthetic genes, confirming its positive regulatory function. Additionally, isothermal titration calorimetry (ITC) and EMSA confirmed that spinosyn A, the main product of the spinosad BGC, served as an effector of ORF-L16. Specifically, it decreased the binding affinity between ORF-L16 and spinosad BGC promoters, thus exerting negative feedback regulation on spinosad biosynthesis. This research enhances our comprehension of spinosad biosynthesis regulation and lays the groundwork for future investigations on transcriptional regulators in S. spinosa.

Deciphering styrene oxide tolerance mechanisms in Gluconobacter oxydans mutant strain

Chemical production wastewater contains large amounts of organic solvents (OSs), which pose a significant threat to the environment. In this study, a 10 g·L−1 styrene oxide tolerant strain with broad-spectrum OSs tolerance was obtained via adaptive laboratory evolution. The mechanisms underlying the high OS tolerance of tolerant strain were investigated by integrating physiological, multi-omics, and genetic engineering analyses. Physiological changes are one of the main factors responsible for the high OS tolerance in mutant strains. Moreover, the P-type ATPase GOX_RS04415 and the LysR family transcriptional regulator GOX_RS04700 were also verified as critical genes for styrene oxide tolerance. The tolerance mechanisms of OSs can be used in biocatalytic chassis cell factories to synthesize compounds and degrade environmental pollutants. This study provides new insights into the mechanisms underlying the toxicological response to OS stress and offers potential targets for enhancing the solvent tolerance of G. oxydans.

Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

New targets of TetR-type regulator SLCG_2919 for controlling lincomycin biosynthesis in Streptomyces lincolnensis.

The transcription factor (TF)-mediated regulatory network controlling lincomycin production in Streptomyces lincolnensis is yet to be fully elucidated despite several types of associated TFs having been reported. SLCG_2919, a tetracycline repressor (TetR)-type regulator, was the first TF to be characterized outside the lincomycin biosynthetic cluster to directly suppress the lincomycin biosynthesis in S. lincolnensis. In this study, improved genomic systematic evolution of ligands by exponential enrichment (gSELEX), an in vitro technique, was adopted to capture additional SLCG_2919-targeted sequences harboring the promoter regions of SLCG_6675, SLCG_4123-4124, SLCG_6579, and SLCG_0139-0140. The four DNA fragments were confirmed by electrophoretic mobility shift assays (EMSAs). Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) showed that the corresponding target genes SLCG_6675 (anthranilate synthase), SLCG_0139 (LysR family transcriptional regulator), SLCG_0140 (beta-lactamase), SLCG_6579 (cytochrome P450), SLCG_4123 (bifunctional DNA primase/polymerase), and SLCG_4124 (magnesium or magnesium-dependent protein phosphatase) in ΔSLCGL_2919 were differentially increased by 3.3-, 4.2-, 3.2-, 2.5-, 4.6-, and 2.2-fold relative to those in the parental strain S. lincolnensis LCGL. Furthermore, the individual inactivation of these target genes in LCGL reduced the lincomycin yield to varying degrees. This investigation expands on the known DNA targets of SLCG_2919 to control lincomycin production and lays the foundation for improving industrial lincomycin yields via genetic engineering of this regulatory network.

LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus.

Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.

β-1,4-Cellobiohydrolase is involved in full expression of phcA, contributing to the feedback loop in quorum sensing of Ralstonia pseudosolanacearum strain OE1-1.

After infecting roots of tomato plants, the gram-negative bacterium Ralstonia pseudosolanacearum strain OE1-1 activates quorum sensing (QS) to induce production of plant cell wall-degrading enzymes, such as β-1,4-endoglucanase (Egl) and β-1,4-cellobiohydrolase (CbhA), via the LysR family transcriptional regulator PhcA and then invades xylem vessels to exhibit virulence. The phcA-deletion mutant (ΔphcA) exhibits neither the ability to infect xylem vessels nor virulence. Compared with strain OE1-1, the egl-deletion mutant (Δegl) exhibits lower cellulose degradation activity, lower infectivity in xylem vessels, and reduced virulence. In this study, we analysed functions of CbhA other than cell wall degradation activity that are involved in the virulence of strain OE1-1. The cbhA-deletion mutant (ΔcbhA) lacked the ability to infect xylem vessels and displayed loss of virulence, similar to ΔphcA, but exhibited less reduced cellulose degradation activity compared with Δegl. Transcriptome analysis revealed that the phcA expression levels in ΔcbhA were significantly lower than in OE1-1, with significantly altered expression of more than 50% of PhcA-regulated genes. Deletion of cbhA led to a significant change in QS-dependent phenotypes, similar to the effects of phcA deletion. Complementation of ΔcbhA with native cbhA or transformation of this mutant with phcA controlled by a constitutive promoter recovered its QS-dependent phenotypes. The expression level of phcA in ΔcbhA-inoculated tomato plants was significantly lower than in strain OE1-1-inoculated plants. Our results collectively suggest that CbhA is involved in the full expression of phcA, thereby contributing to the QS feedback loop and virulence of strain OE1-1.

Identification of a Novel LysR Family Transcriptional Regulator Controlling Acquisition of Sulfur Sources in Acinetobacter baumannii

<sc>l</sc>-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.

Genetic variation in symbiotic islands of natural variant strains of soybean Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens differing in competitiveness and in the efficiency of nitrogen fixation.

Soybean is the most important legume cropped worldwide and can highly benefit from the biological nitrogen fixation (BNF) process. Brazil is recognized for its leadership in the use of inoculants and two strains, Bradyrhizobium japonicum CPAC 15 (=SEMIA 5079) and Bradyrhizobium diazoefficiens CPAC 7 (=SEMIA 5080) compose the majority of the 70 million doses of soybean inoculants commercialized yearly in the country. We studied a collection of natural variants of these two strains, differing in properties of competitiveness and efficiency of BNF. We sequenced the genomes of the parental strain SEMIA 566 of B. japonicum , of three natural variants of this strain (S 204, S 340 and S 370), and compared with another variant of this group, strain CPAC 15. We also sequenced the genome of the parental strain SEMIA 586 of B. diazoefficiens , of three natural variants of this strain (CPAC 390, CPAC 392 and CPAC 394) and compared with the genome of another natural variant, strain CPAC 7. As the main genes responsible for nodulation (nod, noe, nol) and BNF (nif, fix) in soybean Bradyrhizobium are located in symbiotic islands, our objective was to identify genetic variations located in this region, including single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), that could be potentially related to their different symbiotic phenotypes. We detected 44 genetic variations in the B. japonicum strains and three in B. diazoefficiens . As the B. japonicum strains have gone through a longer period of adaptation to the soil, the higher number of genetic variations could be explained by survival strategies under the harsh environmental conditions of the Brazilian Cerrado biome. Genetic variations were detected in genes enconding proteins such as a dephospho-CoA kinase, related to the CoA biosynthesis; a glucosamine-fructose-6-phosphate aminotransferase, key regulator of the hexosamine biosynthetic pathway; a LysR family transcriptional regulator related to nodulation genes; and NifE and NifS proteins, directly related to the BNF process. We suggest potential genetic variations related to differences in the symbiotic phenotypes.

The novel LysR-family transcriptional regulator BvtR is involved in the resistance of Brucella abortus to nitrosative stress, detergents and virulence through the genetic regulation of diverse pathways

Brucella is a facultative intracellular bacterium lacking classical virulence factors; its virulence instead depends on its ability to invade and proliferate within host cells. After entering cells, Brucella rapidly modulates the expression of a series of genes involved in metabolism and immune evasion. Here, a novel LysR-family transcriptional regulator, designated Brucellavirulence-related transcriptional regulator (BvtR), was found to be associated with Brucella abortus virulence. We first successfully constructed a BvtR mutant, ΔbvtR, and a complemented strain, ΔbvtR-Com. Subsequently, we performed cell infection experiments, which indicated that the ΔbvtR strain exhibited similar adhesion, invasion and survival within HeLa cells or RAW264.7 macrophages to those of the wild-type strain. In stress resistance tests, the ΔbvtR strain showed enhanced sensitivity to sodium nitroprusside and sodium dodecyl sulfate, but not to hydrogen peroxide, cumene hydroperoxide, polymyxin B and natural serum. Mouse infection experiments indicated that the virulence of the ΔbvtR strain significantly decreased at 4 weeks post-infection. Finally, we analyzed differentially expressed genes regulated by BvtR with RNA-seq, COG classification and KEGG pathway analysis. Nitrogen metabolism, siderophore biosynthesis and oligopeptide transport were found to be the predominantly altered functions, and key metabolic and regulatory networks were delineated in the ΔbvtR mutant. Thus, we identified a novel Brucella virulence-related regulator, BvtR, and demonstrated that BvtR regulation affects Brucella resistance to killing by sodium nitroprusside and sodium dodecyl sulfate. The differentially expressed genes responding to BvtR are involved in diverse functions or pathways in Brucella, thus, suggesting the breadth of BvtR’s regulatory functions. This study provides novel clues regarding Brucella pathogenesis.

Two LysR Family Transcriptional Regulators, McbH and McbN, Activate the Operons Responsible for the Midstream and Downstream Pathways, Respectively, of Carbaryl Degradation in Pseudomonas sp. Strain XWY-1.

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 was identified by us (Z. Ke, Y. Zhou, W. Jiang, M. Zhang, et al., Appl Environ Microbiol 87:e02970-20, 2021, https://doi.org/10.1128/AEM.02970-20). In this study, we identified McbH and McbN, which activate the mcbIJKLM cluster (responsible for the midstream pathway, from salicylate to gentisate) and the 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 the mcbIJKLM promoter is a 13-bp motif that conforms to the typical characteristics of the 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 revealed the regulatory mechanisms for the midstream and downstream pathways of carbaryl degradation in strain XWY-1 and further our knowledge of (and the size 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 that 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 the 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 LysR-type transcriptional regulator (LTTR) binding sequence affects the binding of McbN to the promoter. These findings help to expand the understanding of the regulatory mechanism of microbial degradation of carbaryl.

PsrA is a novel regulator contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in Serratia marcescens.

Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.

Erratum for Pacheco-Sánchez et al., "DbdR, a New Member of the LysR Family of Transcriptional Regulators, Coordinately Controls Four Promoters in the Thauera aromatica AR-1 3,5-Dihydroxybenzoate Anaerobic Degradation Pathway".

Volume 85, no. 2, e02295-18, 2019, https://doi.org/10.1128/AEM.02295-18. Page 17, Acknowledgments, lines 2 and 3: “… and Ministry of Economy and Competitiveness (BIO2014-54361-R)” should read “and the Spanish Ministry of Economy and Competitiveness (BIO2014-54361-R and BIO2017-82242-R).”

The low-nanomolar 4-nitrobenzoate-responsive repressor PnbX negatively regulates the actinomycete-derived 4-nitrobenzoate-degrading pnb locus.

Nitroaromatic compounds pose severe threats to public health and environmental safety. Nitro group removal via ammonia release is an important strategy for bacterial detoxification of nitroaromatic compounds, such as the conversion of 4-nitrobenzoate (4-NBA) to protocatechuate by the bacterial pnb operon. In contrast to the LysR-family transcriptional regulator PnbR in proteobacteria, the actinomycete-derived pnb locus (4-NBA degradation structural genes) formed an operon with the TetR-family transcriptional regulator gene pnbX, implying that it has a distinct regulatory mechanism. Here, pnbBA from the actinomycete Nocardioides sp. strain LMS-CY was biochemically confirmed to express 4-NBA degradation enzymes, and pnbX was essential for inducible degradation of 4-NBA. Purified PnbX-6His could bind the promoter probe of the pnb locus in vitro, and 4-NBA prevented this binding. 4-NBA could bind PnbX at a 1:1molar ratio with KD = 26.7± 4.2nM. Low-nanomolar levels of 4-NBA induced the transcription of the pnb operon in strain LMS-CY. PnbX bound a palindromic sequence motif (5'-TTACGTTACA-N8 -TGTAACGTAA-3') that encompasses the pnb promoter. This study identified a TetR-family repressor for the actinomycete-derived pnb operon that recognizes 10-8 M 4-NBA as its ligand, implying that nitro group removal of nitroaromatic compounds may be especially important for actinomycetes.

PhcQ mainly contributes to the regulation of quorum sensing-dependent genes, in which PhcR is partially involved, in Ralstonia pseudosolanacearum strain OE1-1.

The gram‐negative plant‐pathogenic β‐proteobacterium Ralstonia pseudosolanacearum strain OE1‐1 produces methyl 3‐hydroxymyristate as a quorum sensing (QS) signal via the methyltransferase PhcB and senses the chemical through the sensor histidine kinase PhcS. This leads to functionalization of the LysR family transcriptional regulator PhcA, regulating QS‐dependent genes responsible for the QS‐dependent phenotypes including virulence. The phc operon consists of phcB, phcS, phcR, and phcQ, with the latter two encoding regulator proteins with a receiver domain and a histidine kinase domain and with a receiver domain, respectively. To elucidate the function of PhcR and PhcQ in the regulation of QS‐dependent genes, we generated phcR‐deletion and phcQ‐deletion mutants. Though the QS‐dependent phenotypes of the phcR‐deletion mutant were largely unchanged, deletion of phcQ led to a significant change in the QS‐dependent phenotypes. Transcriptome analysis coupled with quantitative reverse transcription‐PCR and RNA‐sequencing revealed that phcB, phcK, and phcA in the phcR‐deletion and phcQ‐deletion mutants were expressed at similar levels as in strain OE1‐1. Compared with strain OE1‐1, expression of 22.9% and 26.4% of positively and negatively QS‐dependent genes, respectively, was significantly altered in the phcR‐deletion mutant. However, expression of 96.8% and 66.9% of positively and negatively QS‐dependent genes, respectively, was significantly altered in the phcQ‐deletion mutant. Furthermore, a strong positive correlation of expression of these QS‐dependent genes was observed between the phcQ‐deletion and phcA‐deletion mutants. Our results indicate that PhcQ mainly contributes to the regulation of QS‐dependent genes, in which PhcR is partially involved.

MetR is a molecular adaptor for pneumococcal carriage in the healthy upper airway.

Streptococcus pneumoniae resides in the human upper airway as a commensal but also causes pneumonia, bacteremia, meningitis, and otitis media. It remains unclear how pneumococci adapt to nutritional conditions of various host niches. We here show that MetR, a LysR family transcriptional regulator, serves as a molecular adaptor for pneumococcal fitness, particularly in the upper airway. The metR mutant of strain D39 rapidly disappeared from the nasopharynx but was marginally attenuated in the lungs and bloodstream of mice. RNA-seq and ChIP-seq analyses showed that MetR broadly regulates transcription of the genes involved in methionine synthesis and other functions under methionine starvation. Genetic and biochemical analyses confirmed that MetR is essential for the activation of methionine synthesis but not uptake. Co-infection of influenza virus partially restored the colonization defect of the metR mutant. These results strongly suggest that MetR is particularly evolved for pneumococcal carriage in the upper airway of healthy individuals where free methionine is severely limited, but it becomes dispensable where environmental methionine is relatively more abundant (e.g., inflamed upper airway and sterile sites). To the best of our knowledge, MetR represents the first known regulator particularly for pneumococcal carriage in healthy individuals.

Impact of pmrA on Cronobacter sakazakii planktonic and biofilm cells: A comprehensive transcriptomic study

Cronobacter sakazakii is an emerging opportunistic foodborne pathogen causing rare but severe infections in neonates. Furthermore, the formation of biofilm allows C. sakazakii to persist in different environments. We have demonstrated that the mutator phenotype ascribed to deficiency of the pmrA gene results in more biomass in the first 24 h but less during the post maturation stage (7–14 d) compared with BAA 894. The present study aimed to investigate the regulatory mechanism modulating biofilm formation due to pmrA mutation. The transcriptomic analyses of BAA 894 and s-3 were performed by RNA-sequencing on planktonic and biofilm cells collected at different time points. According to the results, when comparing biofilm to planktonic cells, expression of genes encoding outer membrane proteins, lysozyme, etc. were up-regulated, with LysR family transcriptional regulators, periplasmic proteins, etc. down-regulated. During biofilm formation, cellulose synthase operon genes, flagella-related genes, etc. played essential roles in different stages. Remarkably, pmrA varies the expression of a number of genes related to motility, biofilm formation, and antimicrobial resistance, including srfB, virK, mviM encoding virulence factor, flgF, fliN, etc. encoding flagellar assembly, and marA, ramA, etc. encoding AraC family transcriptional regulators in C. sakazakii. This study provides valuable insights into transcriptional regulation of C. sakazakii pmrA mutant during biofilm formation.

Genomic Analysis of Mesorhizobium loti Strain TONO Reveals Dehalogenases for Bioremediation

Halogenated compounds are extensively utilized in different industrial applications such as pesticides and herbicides and cause severe environmental problems because of their toxicity and persistence. Degradation of these compounds by the biological method is a significant method to reduce these recalcitrant. Mesorhizobium loti is important for nitrogen fixation in legume roots. Up to now, there is no report to indicate M. loti can produce dehalogenase enzymes. Thus, a total of twenty-five genomes of M. loti strains from the National Center for Biotechnology Information (NCBI) were analyzed. These strains notably carry dehalogenase genes and were further investigated. The relative ratio of haloalkane and haloacid dehalogenase type II or L-type from all twenty-five genomes was 26% and 74%, respectively, suggesting type II dehalogenase is common. Surprisingly, only M. loti strain TONO carries four dehalogenases and therefore it was further characterized. The chromosome of M. loti strain TONO contains four haloacid dehalogenase type II genes namely, dehLt1 (MLTONO_2099), dehLt2 (MLTONO_3660), dehLt3 (MLTONO_4143), and dehLt4 (MLTONO_6945), and their corresponding enzymes were designated as DehLt1, DehLt2, DehLt3, and DehLt4, respectively. The only haloalkane dehalogenase gene (MLTONO_4828) was located upstream of the dehLt3 gene and its amino acid share 88% identity with DmlA of Mesorhizobium japonicum MAFF 303099. The putative haloacid permease gene designated as dehrPt (MLTONO_0284) was located downstream of the dehLt1 and its amino acids show 69% identity with haloacid permease of Rhizobium sp. RC1. The gene encoding helix-turn-helix (HTH) motif family DNA-binding protein regulator and LysR family transcriptional regulator genes were also identified, possibly for regulatory functions. The genomic studies as such, have good potential to be screened for new type of dehalogenases based on basic molecular structure and functions analysis

The Escherichia coli azoR gene promoter: A new sensing element for microbial biodetection of trace explosives

Abstract DNT (2,4-dinitrotoluene), a volatile impurity in military grade TNT-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described DNT bioreporter strains, based on a fusion between the Escherichia coli yqjF gene promoter to either a bacterial luxCDABE luminescence gene cassette or to the green fluorescent protein gene gfp. We now present the azoR gene promoter, demonstrated to be strongly induced by DNT, as an alternative sensing element for this compound. An E. coli bioreporter strain harboring a plasmid-borne fusion of the azoR promoter region to Photorhabdus luminescens luxCDABE displayed stronger bioluminescent responses to DNT than the yqjF-based bioreporter. The azoR promoter was induced not directly by DNT but by its degradation products; the most potent inducer was 2,4,5-trihydroxytoluene (THT). This reaction was positively regulated by YhaJ, a member of the LysR family of transcriptional regulators, and was significantly amplified in a yhaK mutant. We also show that DNT degradation in E. coli involves glutathione, which plays a vital role in azoR gene promoter activation.

Genetic Evolution of Mycobacterium abscessus Conferring Clarithromycin Resistance during Long-Term Antibiotic Therapy.

Objectives Clarithromycin is recommended as the core agent for treating M. abscessus infections, which usually calls for at least one year of treatment course, facilitating the development of resistance. This study aimed to identify the underlying mechanism of in vivo development of clarithromycin resistance in M. abscessus clinical isolates. Methods M. abscessus isolates from patients with lung infections during long-term antibiotic therapy were longitudinally collected and sequenced. PFGE DNA fingerprinting was used to confirm the genetic relationships of the isolates. Whole genome comparative analysis was performed to identify the genetic determinants that confer the clarithromycin resistance. Results Three pairs of initially clarithromycin-susceptible and subsequently clarithromycin-resistant M. abscessus isolates were obtained. We found that the clarithromycin-resistant isolates emerged relatively rapidly, after 4–16 months of antibiotic therapy. PFGE DNA fingerprinting showed that the clarithromycin-resistant isolates were identical to the initial clarithromycin-susceptible ones. Whole genome sequencing and bioinformatics analysis identified several genetic alternations in clarithromycin-resistant isolates, including genes encoding efflux pump/transporter, integral component of membrane, and the tetR and lysR family transcriptional regulators. Conclusion We identified genes likely encoding new factors contributing to clarithromycin-resistance phenotype of M. abscessus, which can be useful in prediction of clarithromycin resistance in M. abscessus.