Whole-cell FRET monitoring of transcription factor activities enables functional annotation of signal transduction systems in living bacteria.

Bacteria adapt to their constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor TF-promoter interactions in situ in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular FRET between an unnatural amino acid, l-(7-hydroxycoumarin-4-yl) ethylglycine, which labels TFs with bright fluorescence through genetic encoding (donor fluorophore) and the live cell nucleic acid stain SYTO 9 (acceptor fluorophore). We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems, including one-component (CueR) and two-component systems (BasSR and PhoPQ), in bacteria with high specificity and sensitivity. We demonstrate that robust CouA incorporation and FRET occurrence is achieved in all these regulatory systems based on either the crystal structures of TFs or their simulated structures, if 3D structures of the TFs were unavailable. Furthermore, using CueR and PhoPQ systems as models, we demonstrate that the whole-cell FRET assay is applicable for the identification and validation of complex regulatory circuit and novel modulators of regulatory systems of interest. Finally, we show that the FRET system is applicable for single-cell analysis and monitoring TF activities in Escherichia coli colonizing a Caenorhabditis elegans host. In conclusion, we established a tractable and sensitive TF-promoter binding assay, which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

Gold drugs as colistin adjuvants in the fight against MCR-1 producing bacteria.

The emergence and rapid spread of the mobile colistin resistance gene mcr-1 among bacterial species and hosts significantly challenge the efficacy of "last-line" antibiotic colistin. Previously, we reported silver nitrate and auranofin serve as colistin adjuvants for combating mcr-1-positive bacteria. Herein, we uncovered more gold-based drugs and nanoparticles, and found that they exhibited varying degree of synergisms with colistin on killing mcr-1-positive bacteria. However, pre-activation of the drugs by either glutathione or N-acetyl cysteine, thus releasing and accumulating gold ions, is perquisite for their abilities to substitute zinc cofactor from MCR-1 enzyme. X-ray crystallography and biophysical studies further supported the proposed mechanism. This study not only provides basis for combining gold-based drugs and colistin for combating mcr-1-positive bacterial infections, but also undoubtedly opens a new horizon for metabolism details of gold-based drugs in overcoming antimicrobial resistance.

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation.

The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered due to difficulties of introducing the class-specific, multi-component effectors (Cascade) in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a highly active type I-F Cascade in heterologous bacterial hosts for various genetic exploitations. Using the genetically recalcitrant Pseudomonas species as a paradigm, we show that the transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than both the integrative and plasmid-borne Cas9 systems, and enabled deletion of large fragments such as the 21-kb integrated cassette with efficiency and simplicity. An advanced I-F-λred system was further developed to enable editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an 'all-in-one' I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the programmed mini-CRISPR array in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in 'non-model' bacterial species.

An Isotope-Labeled Single-Cell Raman Spectroscopy Approach for Tracking the Physiological Evolution Trajectory of Bacteria toward Antibiotic Resistance.

Understanding evolution of antibiotic resistance is vital for containing its global spread. Yet our ability to in situ track highly heterogeneous and dynamic evolution is very limited. Here, we present a new single-cell approach integrating D2 O-labeled Raman spectroscopy, advanced multivariate analysis, and genotypic profiling to in situ track physiological evolution trajectory toward resistance. Physiological diversification of individual cells from isogenic population with cyclic ampicillin treatment is captured. Advanced multivariate analysis of spectral changes classifies all individual cells into four subsets of sensitive, intrinsic tolerant, evolved tolerant and resistant. Remarkably, their dynamic shifts with evolution are depicted and spectral markers of each state are identified. Genotypic analysis validates the phenotypic shift and provides insights into the underlying genetic basis. The new platform advances rapid phenotyping resistance evolution and guides evolution control.

Deciphering molecular mechanism of silver by integrated omic approaches enables enhancing its antimicrobial efficacy in E. coli.

Despite the broad-spectrum antimicrobial activities of silver, its internal usage is restricted, owing to the toxicity. Strategies to enhance its efficacy are highly desirable but rely heavily on the understanding of its molecular mechanism of action. However, up to now, no direct silver-targeting proteins have been mined at a proteome-wide scale, which hinders systemic studies on the biological pathways interrupted by silver. Herein, we build up a unique system, namely liquid chromatography gel electrophoresis inductively coupled plasma mass spectrometry (LC-GE-ICP-MS), allowing 34 proteins directly bound by silver ions to be identified in Escherichia coli. By using integrated omic approaches, including metalloproteomics, metabolomics, bioinformatics, and systemic biology, we delineated the first dynamic antimicrobial actions of silver (Ag+) in E. coli, i.e., it primarily damages multiple enzymes in glycolysis and tricarboxylic acid (TCA) cycle, leading to the stalling of the oxidative branch of the TCA cycle and an adaptive metabolic divergence to the reductive glyoxylate pathway. It then further damages the adaptive glyoxylate pathway and suppresses the cellular oxidative stress responses, causing systemic damages and death of the bacterium. To harness these novel findings, we coadministrated metabolites involved in the Krebs cycles with Ag+ and found that they can significantly potentiate the efficacy of silver both in vitro and in an animal model. Our study reveals the comprehensive and dynamic mechanisms of Ag+ toxicity in E. coli cells and offers a novel and general approach for deciphering molecular mechanisms of metallodrugs in various pathogens and cells to facilitate the development of new therapeutics.

Microbiome characteristics and the key biochemical reactions identified on stone world cultural heritage under different climate conditions.

The surfaces of historical stone monuments are visibly covered with a layer of colonizing microorganisms and their degradation products. In this study, a metadata analysis was conducted using the microbial sequencing data available from NCBI database to determine the diversity, biodeterioration potential and functionality of the stone microbiome on important world cultural heritage sites under four different climatic conditions. The retrieved stone microbial community composition in these metagenomes shows a clear association between climate types of the historical monuments and the diversity and taxonomic composition of the stone microbiomes. Shannon diversity values showed that microbial communities on stone monuments exposed to dry climate were more diverse than those under humid ones. In particular, functions associated with photosynthesis and UV resistance were identified from geographical locations under different climate types. The distribution of key microbial determinants responsible for stone deterioration was linked to survival under extreme environmental conditions and biochemical capabilities and reactions. Among them, biochemical reactions of the microbial nitrogen and sulfur cycles were most predominant. These stone-dwelling microbiomes on historical stone monuments were highly diverse and self-sustaining driven by energy metabolism and biomass accumulation. And metabolic products of the internal geomicrobiological nitrogen cycling on these ancient monuments play a unique role in the biodeterioration of stone monuments. These results highlight the significance of identifying the essential microbial biochemical reactions to advance the understanding of stone biodeterioration for protection management.

Harnessing the type I CRISPR-Cas systems for genome editing in prokaryotes.

Genetic analysis is crucial to the understanding, exploitation, and control of microorganisms. The advent of CRISPR-Cas-based genome-editing techniques, particularly those mediated by the single-effector (Cas9 and Cas12a) class 2 CRISPR-Cas systems, has revolutionized the genetics in model eukaryotic organisms. However, their applications in prokaryotes are rather limited, largely owing to the exceptional diversity of DNA homeostasis in microorganisms and severe cytotoxicity of overexpressing these nuclease proteins in certain genotypes. Remarkably, CRISPR-Cas systems belonging to different classes and types are continuously identified in prokaryotic genomes and serve as a deep reservoir for expansion of the CRISPR-based genetic toolkits. ~90% of the CRISPR-Cas systems identified so far belong to the class 1 system which hinges on multi-protein effector complexes for DNA interference. Harnessing these widespread native CRISPR-Cas systems for 'built-in' genome editing represents an emerging and powerful genetic tool in prokaryotes, especially in the genetically recalcitrant non-model species and strains. In this progress review, we introduce the general workflow of this emerging editing platform and summarize its establishment in a growing number of prokaryotes by harnessing the most widespread, diverse type I CRISPR-Cas systems present in their genomes. We also discuss the various factors affecting the success and efficiency of this editing platform and the corresponding solutions.

Detection of synergistic antimicrobial resistance mechanisms in clinical isolates of Pseudomonas aeruginosa from post-operative wound infections.

Infections caused by carbapenem-resistant Pseudomonas aeruginosa are life-threatening due to its synergistic resistance mechanisms resulting in the ineffectiveness of the used antimicrobials. This study aimed to characterize P. aeruginosa isolates for antimicrobial susceptibility, biofilm formation virulence genes, and molecular mechanisms responsible for resistance against various antimicrobials. Out of 700 samples, 91 isolates were confirmed as P. aeruginosa which were further classified into 19 non-multidrug-resistant (non-MDR), 7 multidrug-resistant (MDR), 19 extensively drug-resistant (XDR), and 8 pan drug-resistant (PDR) pulsotypes based on standard Kirby Bauer disc diffusion test and pulse field gel electrophoresis. In M9 minimal media, strong biofilms were formed by the XDR and PDR pulsotypes as compared to the non-MDR pulsotypes. The virulence genes, responsible for the worsening of wounds including LasB, plcH, toxA, and exoU, were detected among all MDR, XDR, and PDR pulsotypes. Carbapenemase activity was phenotypically detected in 45% pulsotypes and the responsible genes were found as blaGES (100%), blaVIM (58%), blaIMP (4%), and blaNDM (4%). Real-time polymerase chain reaction showed the concomitant use of multiple mechanisms such as oprD under-expression, enhanced efflux pump activity, and ampC overexpression in the resistant isolates. Polymyxin is found as the only class left with more than 80% susceptibility among the isolates which is an alarming situation suggesting appropriate measures to be taken including alternative therapies. KEY POINTS: • Multidrug-resistant P. aeruginosa isolates formed stronger biofilms in minimal media. • Only polymyxin antimicrobial was found effective against MDR P. aeruginosa isolates. • Under-expression of oprD and overexpression of ampC were found in resistant isolates.

Zinc excess increases cellular demand for iron and decreases tolerance to copper in Escherichia coli.

Transition metals serve as an important class of micronutrients that are indispensable for bacterial physiology but are cytotoxic when they are in excess. Bacteria have developed exquisite homeostatic systems to control the uptake, storage, and efflux of each of biological metals and maintain a thermodynamically balanced metal quota. However, whether the pathways that control the homeostasis of different biological metals cross-talk and render cross-resistance or sensitivity in the host-pathogen interface remains largely unknown. Here, we report that zinc (Zn) excess perturbs iron (Fe) and copper (Cu) homeostasis in Escherichia coli, resulting in increased Fe and decreased Cu levels in the cell. Gene expression analysis revealed that Zn excess transiently up-regulates Fe-uptake genes and down-regulates Fe-storage genes and thereby increases the cellular Fe quota. In vitro and in vivo protein-DNA binding assays revealed that the elevated intracellular Fe poisons the primary Cu detoxification transcription regulator CueR, resulting in dysregulation of its target genes copA and cueO and activation of the secondary Cu detoxification system CusSR-cusCFBA Supplementation with the Fe chelator 2,2'-dipyridyl (DIP) or with the reducing agent GSH abolished the induction of cusCFBA during Zn excess. Consistent with the importance of this metal homeostatic network in cell physiology, combined metal treatment, including simultaneously overloading cells with both Zn (0.25 mm) and Cu (0.25 mm) and sequestering Fe with DIP (50 µm), substantially inhibited E. coli growth. These results advance our understanding of bacterial metallobiology and may inform the development of metal-based antimicrobial regimens to manage infectious diseases.

Comparative Peptidomic and Metatranscriptomic Analyses Reveal Improved Gamma-Amino Butyric Acid Production Machinery in Levilactobacillus brevis Strain NPS-QW 145 Cocultured with Streptococcus thermophilus Strain ASCC1275 during Milk Fermentation.

The high-gamma-amino butyric acid (GABA)-producing bacterium Levilactobacillus brevis strain NPS-QW 145, along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity), enhances GABA production in milk. However, a mechanistic understanding of how Levilactobacillus brevis cooperates with S. thermophilus to stimulate GABA production has been lacking. Comparative peptidomic and metatranscriptomic analyses were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the coculture. We found that particular peptides hydrolyzed by S. thermophilus ASCC1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was metabolized as the main carbon source by Lb. brevis 145 for ATP production. In the stationary phase, under acidic conditions due to the accumulation of lactic acid produced by S. thermophilus 1275, the expression of genes involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 was induced for GABA production.SIGNIFICANCE A huge market for GABA-rich milk as a dietary therapy for the management of hypertension is anticipated. The novelty of this work lies in applying peptide profiles supported by metatranscriptomics to elucidate (i) the pattern of casein hydrolysis by S. thermophilus 1275, (ii) the supply of peptides and glucose by S. thermophilus 1275 to Lb. brevis 145, (iii) the transportation of peptides in Lb. brevis and the degradation of peptides by this organism, which was reported to be nonproteolytic, and (iv) GABA production by Lb. brevis 145 under acidic conditions. Based on the widely reported contribution of lactic acid bacteria (LAB) and GABA to human health, the elucidation of interactions between the two groups of bacterial communities in the production of GABA-rich milk is important for promoting the development of functional dairy food and may provide new insight into the development of industrial GABA production.

Proteomic Delineation of the ArcA Regulon in Salmonella Typhimurium During Anaerobiosis.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most used models for bacterial pathogenesis and successful infection requires its adaptation to the low oxygen environment in host gastrointestinal tracts. Central to this process is the Arc (aerobic respiratory control) two-component regulatory system that contains a sensor kinase ArcB and a response regulator ArcA. Nevertheless, a comprehensive profile of the ArcA regulon on the proteome level is still lacking in S. Typhimurium. Here we quantitatively profiled Salmonella proteome during anaerobiosis in an arcA-deleting mutant compared with its parental strain. In addition to known processes under its control, notably we found that ArcA represses ethanolamine utilization by directly binding to the promoter region of the eut operon. Furthermore, we found opposing changes of several bacterial genes on the protein and transcript levels in the arcA-deleting mutant including the virulence genes of Salmonella pathogenicity island 1 (SPI-1), thereby indicating potentially prevalent post-transcriptional regulatory mechanisms. Altogether, our study provides important new insights into ArcA-dependent bacterial physiology and virulence during Salmonella anaerobiosis.

Uncoupled Quorum Sensing Modulates the Interplay of Virulence and Resistance in a Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolate Belonging to the MLST550 Clonal Complex.

Pseudomonas aeruginosa is a prevalent and pernicious pathogen equipped with extraordinary capabilities both to infect the host and to develop antimicrobial resistance (AMR). Monitoring the emergence of AMR high-risk clones and understanding the interplay of their pathogenicity and antibiotic resistance is of paramount importance to avoid resistance dissemination and to control P. aeruginosa infections. In this study, we report the identification of a multidrug-resistant (MDR) P. aeruginosa strain PA154197 isolated from a blood stream infection in Hong Kong. PA154197 belongs to a distinctive MLST550 clonal complex shared by two other international P. aeruginosa isolates VW0289 and AUS544. Comparative genome and transcriptome analysis of PA154197 with the reference strain PAO1 led to the identification of a variety of genetic variations in antibiotic resistance genes and the hyperexpression of three multidrug efflux pumps MexAB-OprM, MexEF-OprN, and MexGHI-OpmD in PA154197. Unexpectedly, the strain does not display a metabolic cost and a compromised virulence compared to PAO1. Characterizing its various physiological and virulence traits demonstrated that PA154197 produces a substantially higher level of the P. aeruginosa major virulence factor pyocyanin (PYO) than PAO1, but it produces a decreased level of pyoverdine and displays decreased biofilm formation compared with PAO1. Further analysis revealed that the secondary quorum-sensing (QS) system Pqs that primarily controls the PYO production is hyperactive in PA154197 independent of the master QS systems Las and Rhl. Together, these investigations disclose a unique, uncoupled QS mediated pathoadaptation mechanism in clinical P. aeruginosa which may account for the high pathogenic potentials and antibiotic resistance in the MDR isolate PA154197.

Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853.

BACKGROUND: Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome. RESULTS: Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains. CONCLUSIONS: The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.

Signaling by the heavy-metal sensor CusS involves rearranged helical interactions in specific transmembrane regions.

Two-component systems (TCSs) play important roles in the adaptation of bacteria to stress. Despite their increasingly well understood mechanistic features, it remains poorly understood how TCSs transduce signals across membranes. Here, we use the E. coli Cu/Ag-responsive CusSR TCS as a model to investigate the roles of CusS transmembrane (TM) residues. Proline scanning of TM1 domain led to identification of the T17P, F18P, and S21P variants, which display higher kinase activities relative to wild type. A single point mutation, V202G, in the adjacent TM2 domain specifically suppresses the hyperactivities of these mutants. Disulfide crosslinking analysis demonstrated that T17 and V202 are situated in close proximity, and Cys residues substituted at those two positions form exclusive intramolecular crosslinks when CusS is in the signaling-inactive state. In the signaling-active variant of CusS, however, only intermolecular crosslinking between the two Cys residues could be observed, suggesting that destabilization of an intramolecular constraint and a subsequent rearrangement of helical interactions in this TM region is involved in the activation of CusS. An analogous TM helical interface in the P. aeruginosa heavy metal sensor kinase CzcS is also observed. Together, these results suggested a conserved transmembrane signal transduction mechanism in the heavy metal sensing TCSs.

A novel regulatory circuit to control indole biosynthesis protects Escherichia coli from nitrosative damages during the anaerobic respiration of nitrate.

Indole is a widely distributed microbial secondary metabolite. It mediates a broad range of physiological processes in both its producing and surrounding species. Yet, indole biosynthesis during the anaerobiosis of bacteria remains largely uncharacterized. Here, we find that while indole production is promoted during fermentation and anaerobic respiration of fumarate and trimethylamine N-oxide in E. coli, its biosynthesis is repressed during anaerobic respiration of nitrate especially during exponential growth. We show that expression of the indole biosynthetic operon tnaCAB is repressed under this condition by the two component systems NarXL and NarPQ in the global regulator FNR dependent manner. During stationary growth phase of nitrate respiration, indole biosynthesis is derepressed. However, cellular indole concentration remains low. We demonstrate that this is due to the rapid conversion of indole into mutagenic indole nitrosative derivatives under this condition. Consistent with this, a supplement of exogenous indole during nitrate respiration causes elevated mutation frequencies in E. coli cells lacking the detoxifying efflux genes mdtEF, and ectopic over-expression of tnaAB genes decreases the fitness of E. coli to this physiological condition. Together, these results suggest that indole production is tuned to the bioenergetics activities of E. coli to facilitate its adaptation and fitness.

Biofilms in Endodontics-Current Status and Future Directions.

Microbiota are found in highly organized and complex entities, known as biofilms, the characteristics of which are fundamentally different from microbes in planktonic suspensions. Root canal infections are biofilm mediated. The complexity and variability of the root canal system, together with the multi-species nature of biofilms, make disinfection of this system extremely challenging. Microbial persistence appears to be the most important factor for failure of root canal treatment and this could further have an impact on pain and quality of life. Biofilm removal is accomplished by a chemo-mechanical process, using specific instruments and disinfecting chemicals in the form of irrigants and/or intracanal medicaments. Endodontic research has focused on the characterization of root canal biofilms and the clinical methods to disrupt the biofilms in addition to achieving microbial killing. In this narrative review, we discuss the role of microbial biofilms in endodontics and review the literature on the role of root canal disinfectants and disinfectant-activating methods on biofilm removal.

Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations.

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.

Filamentous prophage Pf4 promotes genetic exchange in Pseudomonas aeruginosa.

Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.

Copper efflux is induced during anaerobic amino acid limitation in Escherichia coli to protect iron-sulfur cluster enzymes and biogenesis.

Adaptation to changing environments is essential to bacterial physiology. Here we report a unique role of the copper homeostasis system in adapting Escherichia coli to its host-relevant environment of anaerobiosis coupled with amino acid limitation. We found that expression of the copper/silver efflux pump CusCFBA was significantly upregulated during anaerobic amino acid limitation in E. coli without the supplement of exogenous copper. Inductively coupled plasma mass spectrometry analysis of the total intracellular copper content combined with transcriptional assay of the P(cusC)-lacZ reporter in the presence of specific Cu(I) chelators indicated that anaerobic amino acid limitation led to the accumulation of free Cu(I) in the periplasmic space of E. coli, resulting in Cu(I) toxicity. Cells lacking cusCFBA and another copper transporter, copA, under this condition displayed growth defects and reduced ATP production during fumarate respiration. Ectopic expression of the Fe-S cluster enzyme fumarate reductase (Frd), or supplementation with amino acids whose biosynthesis involves Fe-S cluster enzymes, rescued the poor growth of ΔcusC cells. Yet, Cu(I) treatment did not impair the Frd activity in vitro. Further studies revealed that the alternative Fe-S cluster biogenesis system Suf was induced during the anaerobic amino acid limitation, and ΔcusC enhanced this upregulation, indicating the impairment of the Fe-S cluster assembly machinery and the increased Fe-S cluster demands under this condition. Taken together, we conclude that the copper efflux system CusCFBA is induced during anaerobic amino acid limitation to protect Fe-S cluster enzymes and biogenesis from the endogenously originated Cu(I) toxicity, thus facilitating the physiological adaptation of E. coli.

Metabolic reprogramming under microaerobic and anaerobic conditions in bacteria.

Oxygen has a great impact on the metabolism and physiology of microorganisms. It serves as the most efficient terminal electron acceptor to drive the energy conservation process of cellular respiration and is required in many biosynthetic reactions. Bacteria encounter oxygen fluctuation and limitation during their growth in both natural ecological niches and in laboratory vessels. In response to oxygen limitation, facultative bacteria undergo substantial metabolic reprogramming to switch from the aerobic respiration to either anaerobic respiration, fermentation, or photosynthesis. Two key factors determine the metabolic pathways bacteria adopt under oxygen deprived microaerobic and anaerobic conditions: maximal energy conservation and redox homeostasis. In this chapter, we first describe how the fulfillment of these two key factors governs the metabolic reprogramming of facultative bacteria and how the process is tightly controlled by several global regulatory factors: FNR, ArcBA, as well as NarL and NarP. We then utilizes fermentation of glycerol, a large surplus byproduct of biodiesel industry, as an example to illustrate how environment, process, and strain based approaches can be exploited to manipulate and engineer the anaerobic metabolic pathways so that desirable fermentation products can be achieved with optimal yield.