An AlgU-Regulated Antisense Transcript Encoded within the Pseudomonas syringae fleQ Gene Has a Positive Effect on Motility.

Production of bacterial flagella is controlled by a multitiered regulatory system that coordinates the expression of 40 to 50 subunits and ordered assembly of these elaborate structures. Flagellar expression is environmentally controlled, presumably to optimize the benefits and liabilities of having these organelles on cell growth and survival. We recently reported a global survey of AlgU-dependent regulation and binding in Pseudomonas syringae pv. tomato DC3000 that included evidence for strong downregulation of many flagellar and chemotaxis motility genes. Here, we returned to those data to look for other AlgU-dependent influences on the flagellar regulatory network. We identified an AlgU-dependent antisense transcript expressed from within the fleQ gene, the master regulator of flagellar biosynthesis in Pseudomonas We tested whether expression of this antisense RNA influenced bacterial behavior and found that it reduces AlgU-dependent downregulation of motility. Importantly, this antisense expression influenced motility only under conditions in which AlgU was expressed. Comparative sequence analysis of the locus containing the antisense transcript's AlgU-dependent promoter in over 300 Pseudomonas genomes revealed sequence conservation in most strains that encode AlgU. This suggests that the antisense transcript plays an important role that is conserved across most of the genus PseudomonasIMPORTANCEPseudomonas syringae is a globally distributed host-specific bacterial pathogen that causes disease in a wide-range of plants. An elaborate gene expression regulation network controls flagellum production, which is important for proper flagellum assembly and a key aspect of certain lifestyle transitions. P. syringae pv. tomato DC3000 uses flagellum-powered motility in the early stages of host colonization and adopts a sessile lifestyle after entering plant tissues, but the regulation of this transition is not understood. Our work demonstrates a link between regulation of motility and global transcriptional control that facilitates bacterial growth and disease in plants. Additionally, sequence comparisons suggest that this regulation mechanism is conserved in most members of the genus Pseudomonas.

Comparative genomics of Pseudomonas syringae pathovar tomato reveals novel chemotaxis pathways associated with motility and plant pathogenicity.

The majority of bacterial foliar plant pathogens must invade the apoplast of host plants through points of ingress, such as stomata or wounds, to replicate to high population density and cause disease. How pathogens navigate plant surfaces to locate invasion sites remains poorly understood. Many bacteria use chemical-directed regulation of flagellar rotation, a process known as chemotaxis, to move towards favorable environmental conditions. Chemotactic sensing of the plant surface is a potential mechanism through which foliar plant pathogens home in on wounds or stomata, but chemotactic systems in foliar plant pathogens are not well characterized. Comparative genomics of the plant pathogen Pseudomonas syringae pathovar tomato (Pto) implicated annotated chemotaxis genes in the recent adaptations of one Pto lineage. We therefore characterized the chemosensory system of Pto. The Pto genome contains two primary chemotaxis gene clusters, che1 and che2. The che2 cluster is flanked by flagellar biosynthesis genes and similar to the canonical chemotaxis gene clusters of other bacteria based on sequence and synteny. Disruption of the primary phosphorelay kinase gene of the che2 cluster, cheA2, eliminated all swimming and surface motility at 21 °C but not 28 °C for Pto. The che1 cluster is located next to Type IV pili biosynthesis genes but disruption of cheA1 has no observable effect on twitching motility for Pto. Disruption of cheA2 also alters in planta fitness of the pathogen with strains lacking functional cheA2 being less fit in host plants but more fit in a non-host interaction.

AlgU Controls Expression of Virulence Genes in Pseudomonas syringae pv. tomato DC3000.

UNLABELLED: Plant-pathogenic bacteria are able to integrate information about their environment and adjust gene expression to provide adaptive functions. AlgU, an extracytoplasmic function (ECF) sigma factor encoded by Pseudomonas syringae, controls expression of genes for alginate biosynthesis and genes involved with resisting osmotic and oxidative stress. AlgU is active while these bacteria are associated with plants, where its presence supports bacterial growth and disease symptoms. We found that AlgU is an important virulence factor for P. syringae pv. tomato DC3000 but that alginate production is dispensable for disease in host plants. This implies that AlgU regulates additional genes that facilitate bacterial pathogenesis. We used transcriptome sequencing (RNA-seq) to characterize the AlgU regulon and chromatin immunoprecipitation sequencing (ChIP-seq) to identify AlgU-regulated promoters associated with genes directly controlled by this sigma factor. We found that in addition to genes involved with alginate and osmotic and oxidative stress responses, AlgU regulates genes with known virulence functions, including components of the Hrp type III secretion system, virulence effectors, and the hrpL and hrpRS transcription regulators. These data suggest that P. syringae pv. tomato DC3000 has adapted to use signals that activate AlgU to induce expression of important virulence functions that facilitate survival and disease in plants. IMPORTANCE: Plant immune systems produce antimicrobial and bacteriostatic conditions in response to bacterial infection. Plant-pathogenic bacteria are adapted to suppress and/or tolerate these conditions; however, the mechanisms controlling these bacterial systems are largely uncharacterized. The work presented here provides a mechanistic explanation for how P. syringae pv. tomato DC3000 coordinates expression of multiple genetic systems, including those dedicated to pathogenicity, in response to environmental conditions. This work demonstrates the scope of AlgU regulation in P. syringae pv. tomato DC3000 and characterizes the promoter sequence regulated by AlgU in these bacteria.

Regulons of three Pseudomonas syringae pv. tomato DC3000 iron starvation sigma factors.

Pseudomonas syringae pv. tomato DC3000 contains genes for 15 sigma factors. The majority are members of the extracytoplasmic function class of sigma factors, including five that belong to the iron starvation subgroup. In this study, we identified the genes controlled by three iron starvation sigma factors. Their regulons are composed of a small number of genes likely to be involved in iron uptake.

An extracytoplasmic function sigma factor-mediated cell surface signaling system in Pseudomonas syringae pv. tomato DC3000 regulates gene expression in response to heterologous siderophores.

The diversity of regulatory systems encoded by bacteria provides an indication of the variety of stresses and interactions that these organisms encounter in nature. We have been investigating how the plant pathogen Pseudomonas syringae pv. tomato DC3000 responds to iron limitation and have focused on the iron starvation (IS) sigma factors to identify regulon members and to explore the mechanistic details of genetic control for this class of regulators. In the study described in this report, we used chromatin immunoprecipitation paired with high-throughput sequencing (ChIP-Seq) to screen the genome for locations associated with binding of the P. syringae IS sigma factor PSPTO_1203. We used multiple methods to demonstrate differential regulation of two genes identified in the ChIP-Seq screen and characterize the promoter elements that facilitate PSPTO_1203-dependent regulation. The genes regulated by PSPTO_1203 encode a TonB-dependent transducer (PSPTO_1206) and a cytoplasmic membrane protein (PSPTO_2145), which is located in the P. syringae pyoverdine cluster. Additionally, we identified siderophores that induce the activity of PSPTO_1203 and used this information to investigate the functional components of the signal transduction cascade.

Characterization of the Fur regulon in Pseudomonas syringae pv. tomato DC3000.

The plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000) is found in a wide variety of environments and must monitor and respond to various environmental signals such as the availability of iron, an essential element for bacterial growth. An important regulator of iron homeostasis is Fur (ferric uptake regulator), and here we present the first study of the Fur regulon in DC3000. Using chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq), 312 chromosomal regions were highly enriched by coimmunoprecipitation with a C-terminally tagged Fur protein. Integration of these data with previous microarray and global transcriptome analyses allowed us to expand the putative DC3000 Fur regulon to include genes both repressed and activated in the presence of bioavailable iron. Using nonradioactive DNase I footprinting, we confirmed Fur binding in 41 regions, including upstream of 11 iron-repressed genes and the iron-activated genes encoding two bacterioferritins (PSPTO_0653 and PSPTO_4160), a ParA protein (PSPTO_0855), and a two-component system (TCS) (PSPTO_3382 to PSPTO_3380).

Recombineering using RecTE from Pseudomonas syringae.

In this report, we describe the identification of functions that promote genomic recombination of linear DNA introduced into Pseudomonas cells by electroporation. The genes encoding these functions were identified in Pseudomonas syringae pv. syringae B728a based on similarity to the lambda Red Exo/Beta and RecET proteins encoded by the lambda and Rac bacteriophages of Escherichia coli. The ability of the pseudomonad-encoded proteins to promote recombination was tested in P. syringae pv. tomato DC3000 using a quantitative assay based on recombination frequency. The results show that the Pseudomonas RecT homolog is sufficient to promote recombination of single-stranded DNA oligonucleotides and that efficient recombination of double-stranded DNA requires the expression of both the RecT and RecE homologs. Additionally, we illustrate the utility of this recombineering system to make targeted gene disruptions in the P. syringae chromosome.

Oligonucleotide recombination: a hidden treasure.

In Swingle et al. we demonstrate that it is possible to use recombineering to direct a variety of changes in wild-type bacterial cells without the addition of phage-encoded proteins. This discovery is potentially applicable to biological engineering in a wide variety of bacterial species. Here we describe key features of oligo recombination as it is currently understood, and propose strategies for expanding the utility of oligo recombination for bioengineering.

Oligonucleotide recombination in Gram-negative bacteria.

This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single-stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any additional phage-encoded functions. Oligo recombination was tested in four genera of Gram-negative bacteria and in all cases evidence for recombination was apparent. The experiments presented here were designed with an eye towards learning to use oligo recombination in order to bootstrap identification and development of phage-encoded recombination systems for recombineering in a wide range of bacteria. The results show that oligo concentration and sequence have the greatest influence on recombination frequency, while oligo length was less important. Apart from the utility of oligo recombination, these findings also provide insights regarding the details of recombination mediated by phage-encoded functions. Establishing that oligos can recombine with bacterial genomes provides a link to similar observations of oligo recombination in archaea and eukaryotes suggesting the possibility that this process is evolutionary conserved.

Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay.

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.

A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties.

Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2'-C-methyl-adenosine and 2'-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2'-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5'-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2'-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2'-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2'-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase.

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells.

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.