Characterization of a novel phage vB_Pae_HB2107-3I that infects Pseudomonas aeruginosa.

Bacteriophages are potential antibiotic substitutes for the treatment of antibiotic resistant bacteria. Here, we report the genome sequences of a double-stranded DNA podovirus vB_Pae_HB2107-3I against clinical multi-drug resistant Pseudomonas aeruginosa. Phage vB_Pae_HB2107-3I remained stable over a wide range of temperatures (37-60 °C) and pH values (pH 4-12). At MOI of 0.01, the latent period of vB_Pae_HB2107-3I was 10 min, and the final titer reached about 8.1 × 109 PFU/mL. The vB_Pae_HB2107-3I genome is 45,929 bp, with an average G + C content of 57%. A total of 72 open reading frames (ORFs) were predicted, of which 22 ORFs have a predicted function. Genome analyses confirmed the lysogenic nature of this phage. Phylogenetic analysis revealed that phage vB_Pae_HB2107-3I was a novel member of Caudovirales infecting P. aeruginosa. The characterization of vB_Pae_HB2107-3I enrich the research on Pseudomonas phages and provide a promising biocontrol agent against P. aeruginosa infections.

Expression of a Phage-Encoded Gp21 Protein Protects Pseudomonas aeruginosa against Phage Infection.

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which has largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection have been identified and characterized in detail. Here, we isolate a phage, vB_Pae_QDWS, capable of infecting Pseudomonas aeruginosa PAO1 and report that its encoded Gp21 protein protects PAO1 against phage infection. Expression of Gp21 regulates bacterial quorum sensing with an inhibitory effect in low cell density and an activation effect in high cell density. By testing the type IV pilus (TFP)-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found to decrease the pilus synthesis. Further, by constructing the TFP synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated that the Gp21 protein could block phage infection via decreasing the TFP-mediated phage adsorption. Gp21 is a novel protein that inhibits phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. IMPORTANCE The majority of the annotated phage genes are currently deposited as "hypothetical protein" with unknown function. Research has revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Conversely, we report a phage encoded protein Gp21 that protects the host against phage infection. The pathways that Gp21 involved in antiphage defense in Pseudomonas aeruginosa PAO1 interfere with quorum sensing and decrease type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage-derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into phage-host evolutionary dynamics.

RetS Regulates Phage Infection in Pseudomonas aeruginosa via Modulating the GacS/GacA Two-Component System.

In Pseudomonas aeruginosa, the complex multisensing regulatory networks RetS-GacS/GacA have been demonstrated to play key roles in controlling the switch between planktonic and sessile lifestyles. However, whether this multisensing system is involved in the regulation of phage infection has not been investigated. Here, we provide a link between the sensors RetS/GacS and infection of phages vB_Pae_QDWS and vB_Pae_W3. Our data suggest that the sensors kinases RetS and GacS in Pseudomonas aeruginosa play opposite regulatory functions on phage infection. Mutation in retS increased phage resistance. Cellular levels of RsmY and RsmZ increased in PaΔretS and were positively correlated with phage resistance. Further analysis demonstrated that RetS regulated phage infection by affecting the type IV pilus (T4P)-mediated adsorption. The regulation of RetS on phage infection depends on the GacS/GacA two-component system and is likely a dynamic process in response to environmental signals. The findings offer additional support for the rapid emergence of phage resistance. IMPORTANCE Our knowledge on the molecular mechanisms behind bacterium-phage interactions remains limited. Our study reported that the complex multisensing regulatory networks RetS-GacS/GacA of Pseudomonas aeruginosa PAO1 play key roles in controlling phage infection. The main observation was that the mutation in RetS could result in increased phage resistance by reducing the type IV pilus-mediated phage adsorption. The bacterial defense strategy is generally applicable to various phages since many P. aeruginosa phages can use type IV pilus as their receptors. The results also suggest that the phage infection is likely to be regulated dynamically, which depends on the environmental stimuli. Reduction of the signals that RetS favors would increase phage resistance. Our study is particularly remarkable for uncovering a signal transduction system that was involved in phage infection, which may help in filling some knowledge gaps in this field.

Characterization of the newly isolated phage Y3Z against multi-drug resistant Cutibacterium acnes.

Cutibacterium acnes (C. acnes) is a symbiotic bacterium that plays an important role in the formation of acn e inflammatory lesions. As a common component of the acne microbiome, C. acnes phages have the potential to make a significant contribution to treating antibiotic-resistant strains of C. acnes. However, little is known about their genetic composition and diversity. In this study, a new lytic phage, Y3Z, infecting C. acne, was isolated and characterized. Electron microscopy analysis revealed this phage is a siphovirus. Phage Y3Z is composed of 29,160 bp with a GC content of 56.32%. The genome contains 40 open reading frames, 17 of which had assigned functions, while no virulence-related genes, antibiotic resistance genes or tRNA were identified. The one-step growth curve showed the burst size was 30 PFU (plaque-forming unit)/cell. And it exhibited tolerance over a broad range of pH and temperature ranges. Phage Y3Z could infect and lyse all C. acnes isolates tested, though the host range of PA6 was restricted to C. acnes. Based on the phylogenetic and comparative genomic analyses, Y3Z may represent a new siphovirus infecting C. acnes. Characterization of Y3Z will enrich our knowledge about the diversity of C. acnes phages and provide a potential arsenal for thetreatment of acne infection.

Complete genome analysis and biological characterization of a novel phage vB_CP_qdyz_P5 with lytic activity against Clostridium perfringens.

Clostridium perfringens, a common foodborne pathogen, exhibit high-stress resistance. The prevailing reliance on antibiotics in the farming industry for its prevention and control has led to increasing concerns over antibiotic residue and bacterial resistance. Bacteriophages that possess specific lytic activity against C. perfringens are of significant interest. Here, a novel C. perfringens phage, named vB_CP_qdyz_P5, was isolated and characterized. The phage displayed high stability at temperatures below 70 °C and pH levels ranging from 4 to 12. Genome analysis revealed that vB_CP_qdyz_P5 has a double-stand DNA of 18,888 bp with a G + C composition of 28.8%. Among the 27 identified opening reading frames (ORFs), eight were found to be functional genes. BLASTn analysis showed that vB_CP_qdyz_P5 is closely related to phage DCp1, with a genome homology coverage of 83%. Phylogenetic analysis indicated that vB_CP_qdyz_P5 may be a novel phage of the family Guelinviridae, Susfortunavirus. This study provides important preliminary information for further research on the potential use of vB_CP_qdyz_P5 in protecting against C. perfringens and maintaining intestinal health.

Phage resistance of Salmonella enterica obtained by transposon Tn5-mediated SefR gene silent mutation.

Salmonella enterica contamination is a primary cause of global food poisoning. Using phages as bactericidal alternatives to antibiotics could confront the issue of drug resistance. However, the problem of phage resistance, especially mutant strains with multiple phage resistance, is a critical barrier to the practical application of phages. In this study, a library of EZ-Tn5 transposable mutants of susceptible host S. enterica B3-6 was constructed. After the infestation pressure of a broad-spectrum phage TP1, a mutant strain with resistance to eight phages was obtained. Analysis of the genome resequencing results revealed that the SefR gene was disrupted in the mutant strain. The mutant strain displayed a reduced adsorption rate of 42% and a significant decrease in swimming and swarming motility, as well as a significantly reduced expression of the flagellar-related FliL and FliO genes to 17% and 36%, respectively. An uninterrupted form of the SefR gene was cloned into vector pET-21a (+) and used for complementation of the mutant strain. The complemented mutant exhibited similar adsorption and motility as the wild-type control. These results suggest that the disrupted flagellar-mediated SefR gene causes an adsorption inhibition, which is responsible for the phage-resistant phenotype of the S. enterica transposition mutant.

Complete genome analysis of the newly isolated Vibrio phage vB_VpP_WS1 of the family Microviridae.

Here, we describe the characterization and genome annotation of the newly isolated lytic Vibrio parahaemolyticus phage vB_VpP_WS1, isolated from sewage samples collected in Qingdao, China. Transmission electron microscopy revealed that vB_VpP_WS1 is about 22 nm in size and that the virions are isometric, likely icosahedral, particles similar to those of members of the Microviridae. The digestion patterns of phage nucleic acids and whole-genome sequencing analysis together revealed that phage vB_VpP_WS1 has a single-stranded DNA genome of 5564 nt. Eight open reading frames were identified, only four of which could be annotated. The proteins of vB_VpP_WS1 displayed low sequence similarity to their homologs encoded by other microviruses. Phylogenetic analysis based on the major capsid protein suggested that vB_VpP_WS1 is a tentative new member of the family Microviridae.

Complete genome analysis of the novel Shewanella phage vB_Sb_QDWS.

We present here the results of the analysis of the complete genome sequence of a potentially temperate phage, vB_Sb_QDWS, which was isolated from wastewater samples collected in Qingdao, China. The genome of phage vB_Sb_QDWS is composed of a double-stranded DNA that is 47,902 bp in length with a G + C content of 63.16%. It is predicted to contain 69 putative protein-encoding genes. Microscopic and genomic analysis showed that vB_Sb_QDWS is a novel phage of the class Siphoviridae.

Study on Volatile Chemicals as Spoilage Indexes of Salmon by HS-SPME-GC-MS Technique during Non-Frozen Storage.

Freshness is the most fundamental and important factor to assess raw fish quality. The purpose of our study was to determine the potential spoilage indexes of salmon during non-frozen storage by using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography-mass spectrometry (GC-MS). More than 300 volatile compounds in salmon were detected when sensory scores declined gradually following the quality changes of salmon at different temperatures. And there were 27 and 31 compounds that showed concentration variations when stored at 4 °C and 25 °C, respectively. Among them, the contents of 1,3-di-tert-butylbenzene, acetic acid, and 3-methyl-1-butanol increased significantly in the later storage period and were in accordance with the salmon's qualities. The present study provides valuable information on the volatile chemical spoilage indexes that are closely related to the freshness of salmon, which may provide an efficient alternative way for quality evaluation of salmon.

Sulfane Sulfur is an intrinsic signal activating MexR-regulated antibiotic resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO1, an opportunistic human pathogen, deploys several strategies to resist antibiotics. It uses multidrug efflux pumps, including the MexAB-OprM pump, for antibiotic resistance, and it also produces hydrogen sulfide (H2 S) that provides some defense against antibiotics. MexR functions as a transcriptional repressor of the mexAB-oprM operon. MexR responds to oxidative stresses caused by antibiotic exposure, and it also displays a growth phase-dependent derepression of the mexAB-oprM operon. However, the intrinsic inducer has not been identified. Here, we report that P. aeruginosa PAO1 produced sulfane sulfur, including glutathione persulfide and inorganic polysulfide, produced from either H2 S oxidation or from L-cysteine metabolism. Sulfane sulfur directly reacted with MexR, forming di- and trisulfide cross-links between two Cys residues, to derepress the mexAB-oprM operon. Levels of cellular sulfane sulfur and mexAB-oprM expression varied during growth, and both reached the maximum during the stationary phase of growth. Thus, self-produced H2 S and sulfane sulfur may facilitate antibiotic resistance via inducing the expression of antibiotic resistance genes.

Cloning and expression of the flavin reductase LuxG from Photobacterium leiognathi YL and its improvement for NADH detection.

In the present study, we aimed to purify and characterize LuxG obtained from Photobacterium leiognathi YL and examine its improvement for NADH detection. To this end, we cloned and expressed the putative luxG gene of P. leiognathi YL in the Escherichia coli BL21 strain. The product of luxG is a flavin reductase that consists of 206 amino acids, corresponding to a subunit molecular mass of ∼26 kDa. Phylogenetic analysis demonstrated that P. leiognathi YL LuxG has a rather distant evolutionary relationship with Frase I of Aliivibrio fischeri and Frp of Vibrio harveyi, but a close evolutionary relationship with Fre from Escherichia coli, which are all enzymes related to oxido-reductase. Further comparison shows that the changes in the functionally conserved sites may contribute to the functional divergence of LuxG and Fre. LuxG could supply reduced flavin mononucleotide (FMN) for bacterial luminescence by catalyzing the oxidation of nicotinamide adenine dinucleotide hydrogen (NADH). Based on this, a coupled pure enzyme bioluminescent system was established and used for NADH detection. The NADH samples with concentrations of 0.1-1 nM were used to validate the linear relationship, and it was found that the logarithmic deviations were less than 3%, which showed more sensitive and stable results than the NADH detection by recombinant E. coli including the exogenously expressed luciferase and intrinsic Fre. Investigation of P. leiognathi YL LuxG would provide a basic understanding of its evolution, and structural and functional properties, which might contribute to the development of a NADH detection kit in the future.

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection.

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1-12 nmol/L) and high (10-500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R2  = 0.9884 and y = 1710× + 4.99 × 105 , R2  = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H-dependent flavin mononucleotide (FMN) reductases.

Strain-level visualized analysis of cold-stressed Vibrio parahaemolyticus based on MALDI-TOF mass fingerprinting.

In this study, strain-level visualized analysis of cold-stressed Vibrio parahaemolyticus based on matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass fingerprinting was investigated. All the peptide mass fingerprinting profiles obtained were analyzed by self-organized map (SOM) and cluster analysis. Our results showed that the peptide mass fingerprinting profiles of V. parahaemolyticus substantially changed under cold stress at strain level. The cold-stressed V. parahaemolyticus strains were distributed to 14 neurons by SOM classification, almost totally different from the controls. This is the first time that so many strains had been chosen to study bacterial cold stress responses, which can help promote an overall understanding to stress responses of cold-stressed strains.

Determination of pyruvic acid concentration using a bioluminescence system from Photobacterium leiognathi.

A novel, highly sensitive and selective bacterial luminescence method for the detection of pyruvic acid (PA) is reported here. This method is based on a reaction system catalyzed by lactate dehydrogenase (LDH) with the bacterial luciferase-FMN:NADH oxidoreductase bioluminescence system in vitro. The reduced nicotinamide adenine dinucleotide (NADH) involved in the LDH reaction system could be quantitatively analyzed by the bioluminescence system. A good linear relationship between the luminescence intensity and pyruvic acid concentration was exhibited within the range of 0.00014-0.001 mol l(-1), and the pyruvic acid detection limit was found to be 8.537 × 10(-5) mol l(-1). This method was successfully applied to the detection of PA in quail serum with a good recovery of over 70%.

MarR family proteins sense sulfane sulfur in bacteria.

Members of the multiple antibiotic resistance regulator (MarR) protein family are ubiquitous in bacteria and play critical roles in regulating cellular metabolism and antibiotic resistance. MarR family proteins function as repressors, and their interactions with modulators induce the expression of controlled genes. The previously characterized modulators are insufficient to explain the activities of certain MarR family proteins. However, recently, several MarR family proteins have been reported to sense sulfane sulfur, including zero-valent sulfur, persulfide (R-SSH), and polysulfide (R-SnH, n ≥ 2). Sulfane sulfur is a common cellular component in bacteria whose levels vary during bacterial growth. The changing levels of sulfane sulfur affect the expression of many MarR-controlled genes. Sulfane sulfur reacts with the cysteine thiols of MarR family proteins, causing the formation of protein thiol persulfide, disulfide bonds, and other modifications. Several MarR family proteins that respond to reactive oxygen species (ROS) also sense sulfane sulfur, as both sulfane sulfur and ROS induce the formation of disulfide bonds. This review focused on MarR family proteins that sense sulfane sulfur. However, the sensing mechanisms reviewed here may also apply to other proteins that detect sulfane sulfur, which is emerging as a modulator of gene regulation.

PqsA mutation-mediated enhancement of phage-mediated combat against Pseudomonas aeruginosa.

Phage therapy is a potential approach in the biocontrol of foodborne pathogens. However, the emergence of phage resistance and the narrow host range of most phage isolates continue to limit the antimicrobial efficacy of phages. Here, we investigated the potential of the pqsA gene, encoding the anthranilate-CoA ligase enzyme, as an adjuvant for phage therapy. The knockout of the pqsA gene significantly enhanced the bactericidal effect of phages vB_Pae_QDWS and vB_Pae_S1 against Pseudomonas aeruginosa. Under phage infection pressure, the growth of the PaΔpqsA was significantly inhibited within 8 h compared to the wild-type PAO1. Furthermore, we found that altering phage adsorption is not how PaΔpqsA responds to phage infection. Although pqsA represents a promising target for enhancing phage killing, it may not be applicable to all phages, such as types vB_Pae_W3 and vB_Pae_TR. Our findings provide new material reserves for the future design of novel phage-based therapeutic strategies.

Shining light on carbon dots: Toward enhanced antibacterial activity for biofilm disruption.

In spite of tremendous efforts dedicated to addressing bacterial infections and biofilm formation, the post-antibiotic ear continues to witness a gap between the established materials and an easily accessible yet biocompatible antibacterial reagent. Here we show carbon dots (CDs) synthesized via a single hydrothermal process can afford promising antibacterial activity that can be further enhanced by exposure to light. By using citric acid and polyethyleneimine as the precursors, the photoluminescence CDs can be produced within a one-pot, one-step hydrothermal reaction in only 2 h. The CDs demonstrate robust antibacterial properties against both Gram-positive and Gram-negative bacteria and, notably, a considerable enhancement of antibacterial effect can be observed upon photo-irradiation. Mechanistic insights reveal that the CDs generate singlet oxygen (1O2) when exposed to light, leading to an augmented reactive oxygen species level. The approach for disruption of biofilms and inhibition of biofilm formation by using the CDs has also been established. Our findings present a potential solution to combat antibacterial resistance and offer a path to reduce dependence on traditional antibiotics.

Quorum sensing autoinducers AHLs protect Shewanella baltica against phage infection.

Quorum sensing (QS) plays an important role in phage-host interactions. Shewanella baltica can't produce the N-acyl-homoserine lactones (AHLs) signal molecules but can eavesdrop on exogenous AHLs through its LuxR receptor. However, no clear evidence exists regarding the involvement of AHLs-mediated QS systems in S. baltica in regulating phage infection. Here, we report that AHLs modulated the phage resistance of S. baltica OS155. Specifically, we characterized a S. baltica phage vB_Sb_QDWS and preliminarily identified that lipopolysaccharide (LPS) is an important receptor for phage vB_Sb_QDWS. AHLs could protect S. baltica against phage infection by decreasing LPS-mediated phage adsorption. The expression of genes galU and tkt, which are essential for LPS synthesis, down-regulated significantly in response to AHLs autoinducers. Our finding confirms the important roles of QS in virus-host interactions and would be helpful to develop novel phage strategies for food spoilage control.

Pseudomonas aeruginosa Resists Phage Infection via Eavesdropping on Indole Signaling.

Phage therapy is challenged by the frequent emergence of bacterial resistance to phages. As an interspecies signaling molecule, indole plays important roles in regulating bacterial behaviors. However, it is unclear whether indole is involved in the phage-bacterium interactions. Here, we report that indole modulated phage resistance of Pseudomonas aeruginosa PAO1. Specifically, we found that the type IV pilus (T4P) acts as an important receptor for P. aeruginosa phages vB_Pae_S1 and vB_Pae_TR, and indole could protect P. aeruginosa against phage infection via decreasing the T4P-mediated phage adsorption. Further investigation demonstrated that indole downregulated the expression of genes pilA, pilB, and pilQ, which are essential for T4P assembly and activity. Indole inhibits phage attacks, but our data suggest that indole functions not through interfering with the AHL-based QS pathway, although las quorum sensing (QS) of P. aeruginosa PAO1 were reported to promote phage infection. Our finding confirms the important roles of indole in virus-host interactions, which will provide important enlightenment in promoting phage therapy for P. aeruginosa infections. IMPORTANCE Our finding is significant with respect to the study of the interactions between phage and host. Although the important roles of indole in bacterial physiology have been revealed, no direct examples of indole participating in phage-host interactions were reported. This study reports that indole could modulate the phage resistance of indole-nonproducing Pseudomonas aeruginosa PAO1 through inhibition of phage adsorption mechanism. Our finding will be significant for guiding phage therapy and fill some gaps in the field of phage-host interactions.

Characterization of the newly isolated Pseudomonas phage vB_Pae_LC3I3.

Here, we report the genome sequence of a double-stranded DNA siphovirus, vB_Pae_LC3I3 infective for P. aeruginosa PA14. Phage vB_Pae_LC3I3 was identified as a linear double-stranded DNA phage of 49,926 bp with 59% G+C content. The vB_Pae_LC3I3 genome contains 78 open reading frames, and the function of 22 ORFs can be predicted. Genome analysis confirmed the lysogenic nature of this phage, which encodes the typical lysogen-related integrase and CI/Cro regulator. One-step growth curve revealed that the latent period of phage vB_Pae_LC3I3 lasted for 30 min. And vB_Pae_LC3I3 showed good temperature stability and pH stability. Based on electron microscopy, phylogenetic, and comparative genomic analyses, this novel Pseudomonas temperate phage represents a novel unassigned siphoviruses cluster. The study of phage vB_Pae_LC3I3 will provide basic information for further research on treatment of P. aeruginosa infections.