PHEIGES: all-cell-free phage synthesis and selection from engineered genomes.

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

Cell-free synthesis of the Salmonella specific broad host range bacteriophage, felixO1.

Phage-based biocontrol of foodborne Salmonella is limited by the requisite use of Salmonella to propagate the phages. This limitation can be circumvented by producing Salmonella phages using a cell-free gene expression system (CFE) with a non-pathogenic chassis. Here, we produce the Salmonella phage felixO1 using an E. coli-based CFE system.

Cell-Free Synthesis and Quantitation of Bacteriophages.

Cell-free transcription-translation (TXTL) enables achieving an ever-growing number of applications, ranging from the rapid characterization of DNA parts to the production of biologics. As TXTL systems gain in versatility and efficacy, larger DNAs can be expressed in vitro extending the scope of cell-free biomanufacturing to new territories. The demonstration that complex entities such as infectious bacteriophages can be synthesized from their genomes in TXTL reactions opens new opportunities, especially for biomedical applications. Over the last century, phages have been instrumental in the discovery of many ground-breaking biotechnologies including CRISPR. The primary function of phages is to infect bacteria. In that capacity, phages are considered an alternative approach to tackling current societal problems such as the rise of antibiotic-resistant microbes. TXTL provides alternative means to produce phages and with several advantages over in vivo synthesis methods. In this chapter, we describe the basic procedures to purify phage genomes, cell-free synthesize phages, and quantitate them using an all-E. coli TXTL system.

Bacteriophage specificity is impacted by interactions between bacteria.

Predators play a central role in shaping community structure, function, and stability. The degree to which bacteriophage predators (viruses that infect bacteria) evolve to be specialists with a single bacterial prey species versus generalists able to consume multiple types of prey has implications for their effect on microbial communities. The presence and abundance of multiple bacterial prey types can alter selection for phage generalists, but less is known about how interactions between prey shape predator specificity in microbial systems. Using a phenomenological mathematical model of phage and bacterial populations, we find that the dominant phage strategy depends on prey ecology. Given a fitness cost for generalism, generalist predators maintain an advantage when prey species compete, while specialists dominate when prey are obligately engaged in cross-feeding interactions. We test these predictions in a synthetic microbial community with interacting strains of Escherichia coli and Salmonella enterica by competing a generalist T5-like phage able to infect both prey against P22vir, an S. enterica-specific phage. Our experimental data conform to our modeling expectations when prey species are competing or obligately mutualistic, although our results suggest that the in vitro cost of generalism is caused by a combination of biological mechanisms not anticipated in our model. Our work demonstrates that interactions between bacteria play a role in shaping ecological selection on predator specificity in obligately lytic bacteriophages and emphasizes the diversity of ways in which fitness trade-offs can manifest. IMPORTANCE: There is significant natural diversity in how many different types of bacteria a bacteriophage can infect, but the mechanisms driving this diversity are unclear. This study uses a combination of mathematical modeling and an in vitro system consisting of Escherichia coli, Salmonella enterica, a T5-like generalist phage, and the specialist phage P22vir to highlight the connection between bacteriophage specificity and interactions between their potential microbial prey. Mathematical modeling suggests that competing bacteria tend to favor generalist bacteriophage, while bacteria that benefit each other tend to favor specialist bacteriophage. Experimental results support this general finding. The experiments also show that the optimal phage strategy is impacted by phage degradation and bacterial physiology. These findings enhance our understanding of how complex microbial communities shape selection on bacteriophage specificity, which may improve our ability to use phage to manage antibiotic-resistant microbial infections.

A ToxIN homolog from Salmonella enterica serotype Enteritidis impairs bacteriophage infection.

AIMS: To determine if the bacteriophage abortive infection system ToxIN is present in foodborne Salmonella and if it protects against infection by bacteriophages specific to enteric bacteria. METHODS AND RESULTS: A set of foodborne Salmonella enteritidis isolates from a 2010 eggshell outbreak was identified via BLASTN (basic local alignment search tool nucleotide) queries as harboring a close homolog of ToxIN, carried on a plasmid with putative mobilization proteins. This homolog was cloned into a plasmid vector and transformed into the laboratory strain Salmonella typhimurium LT2 and tested against a set of Salmonella-specific phages (FelixO1, S16, Sp6, LPST153, and P22 HT105/1 int-201). ToxIN reduced infection by FelixO1, S16, and LPST153 by ∼1-4 log PFU ml-1 while reducing the plaque size of Sp6. When present in LT2 and Escherichia coli MG1655, ToxIN conferred cross-genus protection against phage isolates, which infect both bacteria. Finally, the putative ToxIN plasmid was found in whole-genome sequence contigs of several Salmonella serovars, pathogenic E. coli, and other pathogenic enterobacteria. CONCLUSIONS: Salmonella and E. coli can resist infection by several phages via ToxIN under laboratory conditions; ToxIN is present in foodborne pathogens including Salmonella and Shiga-toxigenic E. coli.

Bacteria intrinsic to Medicago sativa (alfalfa) reduce Salmonella enterica growth in planta.

AIMS: The purpose of this study was to determine whether plant-associated bacteria (PAB) can reduce Salmonella enterica colonization and infection of alfalfa sprouts to reduce the risk of foodborne illness. METHODS: We isolated PAB from alfalfa seeds and sprouts. Monoclonal isolates of the bacteria were obtained and tested for their ability to inhibit Salmonella Typhimurium growth in alfalfa sprouts over 6 days. Genome sequencing and annotation were used to construct draft genomes of the bacteria isolated in this study using Illumina sequencing platform. RESULTS: We observed that a cocktail of five PAB could reduce Salmonella growth in alfalfa sprouts from ∼108 to ∼105 CFU g-1, demonstrating a protective role. Genome sequencing revealed that these bacteria were members of the Pseudomonas, Pantoea, and Priestia genus, and did not possess genes that were pathogenic to plants or animals. CONCLUSIONS: This work demonstrates that PAB can be utilized to reduce pathogen levels in fresh produce, which may be synergistic with other technologies to improve the safety of sprouts and other fresh produce.

Comparative genome analysis of commensal segmented filamentous bacteria (SFB) from turkey and murine hosts reveals distinct metabolic features.

BACKGROUND: Segmented filamentous bacteria (SFB) are intestinal commensal microorganisms that have been demonstrated to induce the innate and adaptive immune responses in mouse and rat hosts. SFB are Gram-positive, spore-forming bacteria that fail to grow optimally under in vitro conditions due to unique metabolic requirements. Recently, SFB have been implicated in improved health and growth outcomes in commercial turkey flocks. To assess the nature and variations in SFB of turkeys and how they may differ from mammalian-associated SFB, the genome of turkey-associated SFB was compared with six representative genomes from murine hosts using an in silico approach. RESULTS: The SFB-turkey genome is 1.6 Mb with a G + C content of 26.14% and contains 1,604 coding sequences (CDS). Comparative genome analyses revealed that all the seven SFB strain possesses a common set of metabolic deficiencies and auxotrophies. Specifically, the inability of all the SFB strains to synthesize most of the amino acids, nucleotides and cofactors, emphasizing the importance of metabolite acquisition from the host intestinal environment. Among the seven SFB genomes, the SFB-turkey genome is the largest and contains the highest number of 1,604 predicted CDS. The SFB-turkey genome possesses cellular metabolism genes that are absent in the rodent SFB strains, including catabolic pathways for sucrose, stachyose, raffinose and other complex glycans. Other unique genes associated with SFB-turkey genome is loci for the biosynthesis of biotin, and degradation enzymes to recycle primary bile acids, both of which may play an important role to help turkey associated SFB survive and secure mutualism with its avian host. CONCLUSIONS: Comparative genomic analysis of seven SFB genomes revealed that each strain have a core set of metabolic capabilities and deficiencies that make these bacteria challenging to culture under ex vivo conditions. When compared to the murine-associated strains, turkey-associated SFB serves as a phylogenetic outgroup and a unique member among all the sequenced strains of SFB. This turkey-associated SFB strain is the first reported non-mammalian SFB genome, and highlights the impact of host specificity and the evolution of metabolic capabilities.

Draft Genome Sequence of "Candidatus Arthromitus" UMNCA01, a Suspected Commensal Isolated from the Gut Microbiome of Commercial Turkey.

"Candidatus Arthromitus" UMNCA01 was recovered from ileal samples of commercial turkey poults and may have probiotic capabilities. The complete genome was determined using the Illumina MiSeq and HiSeq sequencing platforms. The complete genome consists of 1,631,326 bp and has a G+C content of 26.14%, 1,540 coding sequences (CDS), and 37 RNA coding genes.

Nutritional Status According to the Mini Nutritional Assessment Predicts Speed and Degree of Functional Improvement and Discharge Outcomes in Rehabilitation Patients.

This retrospective observational study evaluated the association between nutritional status, functional ability and discharge outcomes. Data from 1430 older rehabilitation patients (43% male, median age 79 years, interquartile range: 74-84) were analyzed. One fifth (20.6%, n = 294) of patients were malnourished on admission to rehabilitation. Three important findings were evident. Firstly, nutritional status on admission to rehabilitation was associated with reduced functional, motor, cognitive and feeding scores on admission and discharge (all P < 0.05). Secondly, malnutrition at admission was associated with significantly slower gains in rehabilitation. Finally, malnutrition at admission was associated with significantly higher odds of a decline in functional ability during admission (OR 3.95; 95% CI: 2.14-7.27), and almost three times greater odds of additional care requirements on discharge (OR: 2.9 ((95% CI: 1.02-8.3). The nutritional status of patients on admission to inpatient rehabilitation is a predictor of both the speed and degree of rehabilitation gains and discharge outcomes.

The rsmS (ybaM) mutation causes bypass suppression of the RsmAB post-transcriptional virulence regulation system in enterobacterial phytopathogens.

Plant cell wall degrading enzymes (PCWDEs) are the primary virulence determinants of soft rotting bacteria such as the potato pathogen, Pectobacterium atrosepticum. The regulation of secondary metabolite (Rsm) system controls production of PCWDEs in response to changing nutrient conditions. This work identified a new suppressor of an rsmB mutation - ECA1172 or rsmS (rsmB suppressor). Mutants defective in rsmB (encoding a small regulatory RNA), show reduced elaboration of the quorum sensing molecule (N-3-oxohexanoyl-homoserine lactone; OHHL) and PCWDEs. However, OHHL and PCWDE production were partially restored in an rsmB, rsmS double mutant. Single rsmS mutants, overproduced PCWDEs and OHHL relative to wild type P. atrosepticum and exhibited hypervirulence in potato. RsmS overproduction also resulted in increased PCWDEs and OHHL. Homology searches revealed rsmS conservation across pathogens such as Escherichia coli (ybaM), Dickeya solani, Klebsiella pneumoniae and Shigella flexneri. An rsmS mutant of Pectobacterium carotovorum ATCC39048 showed bypass of rsmB-dependent repression of PCWDEs and OHHL production. P. carotovorum ATCC39048 produces the ß-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (a carbapenem). Production of the antibiotic was repressed in an rsmB mutant but partially restored in an rsmB, rsmS double mutant. This work highlights the importance of RsmS, as a conserved pleiotropic regulator of virulence and antibiotic biosynthesis.

Evaluation of the impact of a post-hospital discharge Transitional Aged Care Service on frailty, malnutrition and functional ability.

AIM: To investigate the relationship between nutritional status, functional ability and frailty in older adults participating in a 12-week Transitional Aged Care Service program. METHODS: A retrospective analysis of a clinical cohort of older adults aged 65+ years after hospital discharge. At entry into the program and at completion, nutritional status was measured using the Mini Nutritional Assessment (MNA), frailty status was measured using the Groningen Frailty Indicator and functional ability was measured using the Modified Barthel Index (MBI). Demographic data were obtained from electronic medical records. RESULTS: Baseline data were available for 115 participants (mean age = 81.7 (SD =7.9) years; 20.9% classified as malnourished and 89.6% as frail). A positive association was found between nutritional status and frailty (r = 0.298; P = 0.001), and frailty and functional ability (r = 0.204; P = 0.029). Multiple regression analysis, accounting for the cofounders of baseline MNA, MBI, age, gender, length of hospital stay and living situation, found that nutritional status and functional ability were able to indicate the presence of frailty on admission to the program (P = 0.002, P = 0.007, respectively). In those program completers (n = 79), significant improvements were found in nutritional status, frailty and functional ability (P < 0.0005). CONCLUSIONS: Nutrition status, frailty and functional ability are closely and positively related, and should therefore be considered simultaneously in rehabilitation for older adults. A post-hospital transitional program with a multidisciplinary approach significantly improved all three outcomes, suggesting its value in enabling frail older people to remain independent for as long as possible.

Stringent control of FLP recombinase in Escherichia coli.

Site specific recombinases are invaluable tools in molecular biology, and are emerging as powerful recorders of cellular events in synthetic biology. We have developed a stringently controlled FLP recombinase system in Escherichia coli using an arabinose inducible promoter combined with a weak ribosome binding site.

A Comparison of the ATP Generating Pathways Used by S. Typhimurium to Fuel Replication within Human and Murine Macrophage and Epithelial Cell Lines.

The metabolism of S. Typhimurium within infected host cells plays a fundamental role in virulence since it enables intracellular proliferation and dissemination and affects the innate immune response. An essential requirement for the intracellular replication of S. Typhimurium is the need to regenerate ATP. The metabolic route used to fulfil this requirement is the subject of the present study. For infection models we used human and murine epithelial and macrophage cell lines. The epithelial cell lines were mICc12, a transimmortalised murine colon enterocyte cell line that shows many of the characteristics of a primary epithelial cell line, and HeLa cells. The model macrophage cell lines were THP-1A human monocyte/macrophages and RAW 264.7 murine macrophages. Using a mutational approach combined with an exometabolomic analysis, we showed that neither fermentative metabolism nor anaerobic respiration play major roles in energy generation in any of the cell lines studied. Rather, we identified overflow metabolism to acetate and lactate as the foremost route by which S. Typhimurium fulfils its energy requirements.

Toward Network Biology in E. coli Cell.

E. coli has been a critically important model research organism for more than 50 years, particularly in molecular biology. In 1997, the E. coli draft genome sequence was published. Post-genomic techniques and resources were then developed that allowed E. coli to become a model organism for systems biology. Progress made since publication of the E. coli genome sequence will be summarized.

Mep72, a metzincin protease that is preferentially secreted by biofilms of Pseudomonas aeruginosa.

In this work, we compared the profile of proteins secreted by planktonic and biofilm cultures of Pseudomonas aeruginosa using two-dimensional difference gel electrophoresis (2D-DiGE). This revealed that a novel metzincin protease, Mep72, was secreted during biofilm growth. Subsequent Western blotting and reverse transcription-PCR (RT-PCR) analyses demonstrated that Mep72 was expressed only during biofilm growth. Mep72 has a tridomain structure comprised of a metzincin protease-like domain and two tandem carbohydrate-binding domains. Unlike the only other metzincin (alkaline protease; AprA) in P. aeruginosa, Mep72 is secreted through the type II pathway and undergoes processing during export. During this processing, the metzincin domain is liberated from the carbohydrate-binding domains. This processing may be self-catalyzed, since purified Mep72 autodegraded in vitro. This autodegradation was retarded in the presence of alginate (an extracellular matrix component of many P. aeruginosa biofilms). The expression of full-length mep72 in Escherichia coli was toxic. However, this toxicity could be alleviated by coexpression of mep72 with the adjacent gene, bamI. Mep72 and BamI were found to form a protein-protein complex in vitro. 2D-DiGE revealed that the electrophoretic mobility of several discrete protein spots was altered in the biofilm secretome of an mep72 mutant, including type III secretion proteins (PopD, PcrV, and ExoS) and a flagellum-associated protein (FliD). Mep72 was found to bind directly to ExoS and PcrV and to affect the processing of these proteins in the biofilm secretome. We conclude that Mep72 is a secreted biofilm-specific regulator that affects the processing of a very specific subset of virulence factors.

Nutritional and metabolic requirements for the infection of HeLa cells by Salmonella enterica serovar Typhimurium.

Salmonella is the causative agent of a spectrum of human and animal diseases ranging from gastroenteritis to typhoid fever. It is a food--and water--borne pathogen and infects via ingestion followed by invasion of intestinal epithelial cells and phagocytic cells. In this study we employed a mutational approach to define the nutrients and metabolic pathways required by Salmonella enterica serovar Typhimurium during infection of a human epithelial cell line (HeLa). We deleted the key glycolytic genes, pfkA and pfkB to show that S. Typhimurium utilizes glycolysis for replication within HeLa cells; however, glycolysis was not absolutely essential for intracellular replication. Using S. Typhimurium strains deleted for genes encoding components of the phosphotransferase system and glucose transport, we show that glucose is a major substrate required for the intracellular replication of S. Typhimurium in HeLa cells. We also deleted genes encoding enzymes involved in the utilization of gluconeogenic substrates and the glyoxylate shunt and show that neither of these pathways were required for intracellular replication of S. Typhimurium within HeLa cells.

Surface swarming motility by Pectobacterium atrosepticum is a latent phenotype that requires O antigen and is regulated by quorum sensing.

We describe a previously cryptic phenotype associated with the opportunistic phytopathogen Pectobacterium atrosepticum (Pca): surface swarming. We found that when Pca was spotted onto plates containing <0.5% (w/v) agar, the culture produced copious amounts of extracellular matrix material containing highly motile cells. Once produced, this 'slime layer' spread rapidly across the plate either as an advancing front or as tendrils. Transposon mutagenesis was used to identify mutants that were affected in swarming. Hypo-swarmer mutants mostly carried insertions in a horizontally acquired island (HAI5), which encodes a cluster of genes involved in O antigen biosynthesis. Hyper-swarmer mutants mostly carried insertions in hexY, a known antagonist of the class I flagellar master regulator, FlhD4C2. In addition, we found that the nucleoid protein, histone-like nuclear structuring protein 2 (H-NS2), also regulated swarming behaviour. A mutant in which hns2 was overexpressed displayed a hyper-swarming phenotype, whereas a mutant in which the hns2 ORF was inactivated had a hypo-swarming phenotype. Swarming was also regulated by quorum sensing (QS) and by the carbon source being utilized. We show, using a range of epistasis experiments, that optimal swarming requires both motility and O antigen biosynthesis, and that H-NS2 and QS both promote swarming through their effects on motility.

Virulence in Pectobacterium atrosepticum is regulated by a coincidence circuit involving quorum sensing and the stress alarmone, (p)ppGpp.

Pectobacterium atrosepticum (Pca) is a Gram-negative phytopathogen which causes disease by secreting plant cell wall degrading exoenzymes (PCWDEs). Previous studies have shown that PCWDE production is regulated by (i) the intercellular quorum sensing (QS) signal molecule, 3-oxo-hexanoyl-l-homoserine lactone (OHHL), and (ii) the intracellular 'alarmone', (p)ppGpp, which reports on nutrient limitation. Here we show that these two signals form an integrated coincidence circuit which ensures that metabolically costly PCWDE synthesis does not occur unless the population is simultaneously quorate and nutrient limited. A (p)ppGpp null ΔrelAΔspoT mutant was defective in both OHHL and PCWDE production, and nutritional supplementation of wild type cultures (which suppresses (p)ppGpp production) also suppressed OHHL and PCWDE production. There was a substantial overlap in the transcriptome of a (p)ppGpp deficient relA mutant and of a QS defective expI (OHHL synthase) mutant, especially with regards to virulence-associated genes. Random transposon mutagenesis revealed that disruption of rsmA was sufficient to restore PCWDE production in the (p)ppGpp null strain. We found that the ratio of RsmA protein to its RNA antagonist, rsmB, was modulated independently by (p)ppGpp and QS. While QS predominantly controlled virulence by modulating RsmA levels, (p)ppGpp exerted regulation through the modulation of the RsmA antagonist, rsmB.

Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Applications of small molecule activators and inhibitors of quorum sensing in Gram-negative bacteria.

Quorum sensing is a form of intercellular communication used by many species of bacteria that facilitates concerted interactions between the cells comprising a population. The phenotypes regulated by quorum sensing are extremely diverse, with many having a significant impact upon healthcare, agriculture, and the environment. Consequently there has been significant interest in developing methods to manipulate this signalling process and recent years have witnessed significant theoretical and practical developments. A wide range of small molecule modulators of quorum sensing systems has been discovered, providing an expansive chemical toolbox for the study and modulation of this signalling mechanism. In this review, a selection of recent case studies which illustrate the value of both activators and inhibitors of quorum sensing in Gram-negative bacteria are discussed.