Electrotransformation of Foodborne Pathogen Cronobacter sakazakii by a Simple Method.

Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.

Structural basis for diguanylate cyclase activation by its binding partner in Pseudomonas aeruginosa.

Cyclic-di-guanosine monophosphate (c-di-GMP) is an important effector associated with acute-chronic infection transition in Pseudomonas aeruginosa. Previously, we reported a signaling network SiaABCD, which regulates biofilm formation by modulating c-di-GMP level. However, the mechanism for SiaD activation by SiaC remains elusive. Here we determine the crystal structure of SiaC-SiaD-GpCpp complex and revealed a unique mirror symmetric conformation: two SiaD form a dimer with long stalk domains, while four SiaC bind to the conserved motifs on the stalks of SiaD and stabilize the conformation for further enzymatic catalysis. Furthermore, SiaD alone exhibits an inactive pentamer conformation in solution, demonstrating that SiaC activates SiaD through a dynamic mechanism of promoting the formation of active SiaD dimers. Mutagenesis assay confirmed that the stalks of SiaD are necessary for its activation. Together, we reveal a novel mechanism for DGC activation, which clarifies the regulatory networks of c-di-GMP signaling.

Iron facilitates the RetS-Gac-Rsm cascade to inversely regulate protease IV (piv) expression via the sigma factor PvdS in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces several proteases, such as an elastase (LasB protease), a LasA protease, and protease IV (PIV), which are thought as significant virulence factors during infection. Regulators of LasA and LasB expression have been identified and well characterized; however, the molecular details of this regulation of protease IV (PIV) remained largely unknown. Here, we describe the interaction between protease IV and the RetS/Rsm signalling pathway, which plays a central role in controlling the production of multiple virulence factors and the switch from planktonic to biofilm lifestyle. We show that the expression of piv was reduced in ΔretS or ΔrsmA strain grown under restrictive conditions but was induced in ΔretS or ΔrsmA mutant grown under rich conditions as compared with wild-type parent. We compare the expression of piv under various conditions and found that iron facilitates RetS/Rsm system to lead this inverse regulation. Moreover, we reveal that the RetS/Rsm pathway regulates PIV production dependent on the alternative sigma factor PvdS. Collectively, this study extends the understanding of the RetS/Rsm regulatory cascade in response to environmental signals and provides insights into how P. aeruginosa adapts to the complex conditions.

A novel c-di-GMP signal system regulates biofilm formation in Pseudomonas aeruginosa.

The bacterial second messenger cyclic-di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. The human pathogen Pseudomonas aeruginosa encodes 17 diguanylate cyclase (DGCs) proteins which are required for c-di-GMP synthesis. Therefore, the c-di-GMP regulatory system in P. aeruginosa is highly sophisticated. SiaD, one of the DGC enzymes, is co-transcribed with SiaA/B/C and has been shown to be essential for bacterial aggregate formation in response to environmental stress. However, the detailed function of this operon remains unknown. In our recent paper (Chen et al., doi: 10.15252/embj.2019103412), we have demonstrated that the siaABCD operon encodes a signaling network that regulates biofilm and aggregate formation by modulating the enzymatic activity of SiaD. Among this signaling system, SiaC interaction with SiaD promotes the diguanylate cyclase activity of SiaD and subsequently facilities the intracellular c-di-GMP synthesis; SiaB is a unique protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD. This report unveils a novel signaling system that controls biofilm formation, which may provide a potential target for developing antimicrobial drugs.

The SiaA/B/C/D signaling network regulates biofilm formation in Pseudomonas aeruginosa.

Bacterial cyclic-di-GMP (c-di-GMP) production is associated with biofilm development and the switch from acute to chronic infections. In Pseudomonas aeruginosa, the diguanylate cyclase (DGC) SiaD and phosphatase SiaA, which are co-transcribed as part of a siaABCD operon, are essential for cellular aggregation. However, the detailed functions of this operon and the relationships among its constituent genes are unknown. Here, we demonstrate that the siaABCD operon encodes for a signaling network that regulates SiaD enzymatic activity to control biofilm and aggregates formation. Through protein-protein interaction, SiaC promotes SiaD diguanylate cyclase activity. Biochemical and structural data revealed that SiaB is an unusual protein kinase that phosphorylates SiaC, whereas SiaA phosphatase can dephosphorylate SiaC. The phosphorylation state of SiaC is critical for its interaction with SiaD, which will switch on or off the DGC activity of SiaD and regulate c-di-GMP levels and subsequent virulence phenotypes. Collectively, our data provide insights into the molecular mechanisms underlying the modulation of DGC activity associated with chronic infections, which may facilitate the development of antimicrobial drugs.

A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition.

The type VI secretion system (T6SS) is widely distributed in Gram-negative bacteria, whose function is known to translocate substrates to eukaryotic and prokaryotic target cells to cause host damage or as a weapon for interbacterial competition. Pseudomonas aeruginosa encodes three distinct T6SS clusters (H1-, H2-, and H3-T6SS). The H1-T6SS-dependent substrates have been identified and well characterized; however, only limited information is available for the H2- and H3-T6SSs since relatively fewer substrates for them have yet been established. Here, we obtained P. aeruginosa H2-T6SS-dependent secretomes and further characterized the H2-T6SS-dependent copper (Cu2+)-binding effector azurin (Azu). Our data showed that both azu and H2-T6SS were repressed by CueR and were induced by low concentrations of Cu2+. We also identified the Azu-interacting partner OprC, a Cu2+-specific TonB-dependent outer membrane transporter. Similar to H2-T6SS genes and azu, expression of oprC was directly regulated by CueR and was induced by low Cu2+. In addition, the Azu-OprC-mediated Cu2+ transport system is critical for P. aeruginosa cells in bacterial competition and virulence. Our findings provide insights for understanding the diverse functions of T6SSs and the role of metal ions for P. aeruginosa in bacteria-bacteria competition.

Glutathione Activates Type III Secretion System Through Vfr in Pseudomonas aeruginosa.

Glutathione (GSH) is the most abundant antioxidant in all living organisms. Previously, we have shown that a deletion mutant in the glutathione synthetase gene (ΔgshB) decreases the expression of type III secretion system (T3SS) genes of Pseudomonas aeruginosa. However, the mechanism remains elusive. In this study, a comprehensive transcriptomic analysis of the GSH-deficient mutant ΔgshAΔgshB was used to elucidate the role of GSH in the pathogenesis of P. aeruginosa. The data show that the expression of genes in T3SS, type VI secretion system (T6SS) and some regulatory genes were impaired. ΔgshAΔgshB was attenuated in a mouse model of acute pneumonia, swimming and swarming motilities, and biofilm formation. Under T3SS inducing conditions, GSH enhanced the expression of T3SS in both wild-type PAO1 and ΔgshAΔgshB, but not in Δvfr. Genetic complementation of Δvfr restored the ability of GSH to induce the expression of T3SS genes. Site-directed mutagenesis based substitution of cysteine residues with alanine in Vfr protein abolished the induction of T3SS genes by GSH, confirming that GSH regulates T3SS genes through Vfr. Exposure to H2O2 decreased free thiol content on Vfr, indicating that the protein was sensitive to redox modification. Importantly, GSH restored the oxidized Vfr to reduced state. Collectively, these results suggest that GSH serves as an intracellular redox signal sensed by Vfr to upregulate T3SS expression in P. aeruginosa. Our work provides new insights into the role of GSH in P. aeruginosa pathogenesis.

Oligoribonuclease Contributes to Tolerance to Aminoglycoside and ß-Lactam Antibiotics by Regulating KatA in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen and is intrinsically resistant to a variety of antibiotics. Oligoribonuclease (Orn) is a 3'-to-5' exonuclease that degrades nanoRNAs. The Orn controls biofilm formation by influencing the homeostasis of cyclic-di-GMP. Previously, we demonstrated that Orn contributes to the tolerance of P. aeruginosa to fluoroquinolone antibiotics by affecting the production of pyocins. In this study, we found that mutation in the orn gene reduces bacterial tolerance to aminoglycoside and ß-lactam antibiotics, which is mainly due to a defective response to oxidative stresses. The major catalase KatA is downregulated in the orn mutant, and overexpression of the katA gene restores the bacterial tolerance to oxidative stresses and the antibiotics. We further demonstrated that Orn influenced the translation of the katA mRNA and narrowed down the region in the katA mRNA that is involved in the regulation of its translation. Therefore, our results revealed a novel role of the Orn in bacterial tolerance to oxidative stresses as well as aminoglycoside and ß-lactam antibiotics.

Molecular insights into the master regulator CysB-mediated bacterial virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major pathogen that causes serious acute and chronic infections in humans. The type III secretion system (T3SS) is an important virulence factor that plays essential roles in acute infections. However, the regulatory mechanisms of T3SS are not fully understood. In this study, we found that the deletion of cysB reduced the T3SS gene expression and swarming motility but enhanced biofilm formation. In a mouse acute pneumonia model, mutation of cysB decreased the average bacterial load compared to that of the wild-type strain. Further experiments demonstrated that CysB contributed to the reduced T3SS gene expression and bacterial pathogenesis by directly regulating the sensor kinase RetS. We also performed crystallographic studies of PaCysB. The overall fold of PaCysB NTD domain is similar to other LysR superfamily proteins and structural superposition revealed one possible DNA-binding model for PaCysB. Structural comparison also revealed great flexibility of the PaCysB RD domain, which may play an important role in bending and transcriptional regulation of target DNA. Taken together, these results expand our current understanding of the complex regulatory networks of T3SS and RetS pathways. The crystal structure of CysB provides new insights for studying the function of its homologs in other bacterial species.

Multilocus sequence typing and variations in the oprD gene of Pseudomonas aeruginosa isolated from a hospital in China.

OBJECTIVES: To provide information about the genetic relationships and mechanism underlying carbapenem resistance in Pseudomonas aeruginosa clinical isolates of a hospital in China. MATERIALS AND METHODS: One hundred and sixty P. aeruginosa strains were isolated from a hospital in China. Susceptibility to 14 antimicrobial agents was determined by antimicrobial susceptibility testing. Multilocus sequence typing was used to characterize the genetic backgrounds of these clinical isolates. Forty-five strains were randomly selected for further evaluation of their carbapenem resistance mechanism. Their oprD gene was compared with the PAO1 sequence. RESULTS: Multilocus sequence typing analysis demonstrated that these isolates were highly diverse; 68 sequence types were identified, of which 28 were novel sequence types. Polygenic and eBURST analysis demonstrated genetically similar clones with dissimilar resistance profiles. Among the 45 randomly selected strains associated with carbapenem resistance, 2 were metallo ß-lactamase producers; all the 45 strains were not AmpC overproducers. Sequence analysis revealed a high diversity in the oprD sequences among isolates. Strains susceptible to imipenem and meropenem with shortened L7 and L8 loops in oprD were the major strain types observed in this hospital. CONCLUSION: This study indicated that oprD provided the main mechanism for carbapenem resistance. The shortened L7 and L8 loops are responsible for carbapenem susceptibility.

Pseudomonas aeruginosa Oligoribonuclease Contributes to Tolerance to Ciprofloxacin by Regulating Pyocin Biosynthesis.

Bacterial oligoribonuclease (Orn) is a conserved 3'-to-5' exonuclease. In Pseudomonas aeruginosa, it has been demonstrated that Orn plays a major role in the hydrolysis of pGpG, which is required for cyclic-di-GMP homeostasis. Meanwhile, Orn is involved in the degradation of nanoRNAs, which can alter global gene expression by serving as transcription initiation primers. Previously, we found that Orn is required for the type III secretion system and pathogenesis of P. aeruginosa, indicating a role of Orn in the bacterial response to environmental stimuli. Here we report that Orn is required for the tolerance of P. aeruginosa to ciprofloxacin. Transcriptome analysis of an orn mutant revealed the upregulation of pyocin biosynthesis genes. Mutation of genes involved in pyocin biosynthesis in the background of an orn mutant restored bacterial tolerance to ciprofloxacin. We further demonstrate that the upregulation of pyocin biosynthesis genes is due to RecA-mediated autoproteolysis of PrtR, which is the major negative regulator of pyocin biosynthesis genes. In addition, the SOS response genes were upregulated in the orn mutant, indicating a DNA damage stress. Therefore, our results revealed a novel role of Orn in bacterial tolerance to ciprofloxacin.

The P-Type ATPase PA1429 Regulates Quorum-Sensing Systems and Bacterial Virulence.

Pseudomonas aeruginosa is becoming an increasingly prevalent pathogen, capable of causing numerous life threatening infections in immunocompromised patients. The three hierarchically arranged quorum sensing (QS) systems, namely las, rhl, and pqs play key roles in coordinating virulence expression in P. aeruginosa. However, the regulatory mechanisms of the pqs system have not been fully elucidated. To identify new genes involved in synthesis of the Pseudomonas quinolone signal (PQS), a transposon mutagenesis library was constructed. PA1429 was found to inhibit PQS biosynthesis. The PA1429 deletion mutant also exhibited increased bacterial motility, biofilm formation, and virulence in a mouse model of acute lung infection. Interestingly, it also displayed reduced tolerance to oxidative stress. Mutated pqsH in the PA1429 deletion background restored bacterial susceptibility to H2O2. In addition, our data showed that PA1429 repressed the expression of las and rhl systems. Overall, these results provide new insights into the complex regulatory networks of quorum-sensing and virulence expression in P. aeruginosa.

Oligoribonuclease is required for the type III secretion system and pathogenesis of Pseudomonas aeruginosa.

Oligoribonuclease (Orn) is a 3' to 5' exonuclease that degrades nanoRNAs, which can serve as primers for transcription initiation at a significant fraction of promoters. One of Orn's substrates, pGpG inhibits the enzymatic activity of EAL-domain containing phosphodiesterases (PDEs), thereby increasing intracellular cyclic-di-GMP (c-di-GMP) level. Here, we found that an orn mutant of Pseudomonas aeruginosa displayed reduced cytotoxicity, which was mainly due to deficient type III secretion system (T3SS). Given the importance of T3SS in pathogenicity, we examined the bacterial virulence in a mouse acute pneumonia model and found that the Δorn mutant was highly attenuated compared to the wild type PA14 strain. Overexpression of an EAL domain-containing PDE reduced the c-di-GMP level as well as biofilm formation in the Δorn mutant. However, no effect was observed on the expression of T3SS genes, suggesting that increased c-di-GMP level is not the solely cause of defective T3SS in the Δorn mutant. Overall, our results demonstrated an essential role of Orn in the expression of T3SS as well as pathogenesis of P. aeruginosa.

SuhB is a regulator of multiple virulence genes and essential for pathogenesis of Pseudomonas aeruginosa.

UNLABELLED: During initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa. IMPORTANCE: A variety of bacterial pathogens, such as Pseudomonas aeruginosa, cause acute and chronic infections in humans. During infections, pathogens produce different sets of virulence genes for colonization, tissue damage, and dissemination and for countering host immune responses. Complex regulatory networks control the delicate tuning of gene expression in response to host environments to enable the survival and growth of invading pathogens. Here we identified suhB as a critical gene for the regulation of virulence factors in P. aeruginosa. The expression of suhB was upregulated during acute infection in an animal model, and mutation of suhB rendered P. aeruginosa avirulent. Moreover, we demonstrate that SuhB is required for the activation of virulence factors associated with acute infections while suppressing virulence factors associated with chronic infections. Our report provides new insights into the multilayered regulatory network of virulence genes in P. aeruginosa.

Construction of Escherichia coli strains producing L-serine from glucose.

L-Serine is usually produced from glycine. We have genetically engineered Escherichia coli to produce L-serine from glucose intracellularly. D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) in E. coli catalyzes the first committed step in L-serine formation but is inhibited by L-serine. To overcome this feedback inhibition, both the His(344) and Asn(346) residues of PGDH were converted to alanine and the mutated PGDH (PGDH(dr)) became insensitive to L-serine. However, overexpression of PGDH(dr) gave no significant increase of L-serine accumulation but, when L-serine deaminase genes (sdaA, sdaB and tdcG) were deleted, serine accumulated: (1) deletion of sdaA gave up to 0.03 mmol L-serine/g; (2) deletion of both sdaA and sdaB accumulated L-serine up to 0.09 mmol/g; and (3) deletion of sdaA, sdaB and tdcG gave up to 0.13 mmol L-serine/g cell dry wt.