Identification of a Novel Pyruvyltransferase Using 13C Solid-State Nuclear Magnetic Resonance To Analyze Rhizobial Exopolysaccharides.

The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes (exoZ, exoH, SMb20810, SMb21188, and SMa1016) and a putative pyruvyltransferase (wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE. Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.

Implementation of deep learning-based auto-segmentation for radiotherapy planning structures: a workflow study at two cancer centers.

PURPOSE: We recently described the validation of deep learning-based auto-segmented contour (DC) models for organs at risk (OAR) and clinical target volumes (CTV). In this study, we evaluate the performance of implemented DC models in the clinical radiotherapy (RT) planning workflow and report on user experience. METHODS AND MATERIALS: DC models were implemented at two cancer centers and used to generate OAR and CTVs for all patients undergoing RT for a central nervous system (CNS), head and neck (H&N), or prostate cancer. Radiation Therapists/Dosimetrists and Radiation Oncologists completed post-contouring surveys rating the degree of edits required for DCs (1 = minimal, 5 = significant) and overall DC satisfaction (1 = poor, 5 = high). Unedited DCs were compared to the edited treatment approved contours using Dice similarity coefficient (DSC) and 95% Hausdorff distance (HD). RESULTS: Between September 19, 2019 and March 6, 2020, DCs were generated on approximately 551 eligible cases. 203 surveys were collected on 27 CNS, 54 H&N, and 93 prostate RT plans, resulting in an overall survey compliance rate of 32%. The majority of OAR DCs required minimal edits subjectively (mean editing score ≤ 2) and objectively (mean DSC and 95% HD was ≥ 0.90 and ≤ 2.0 mm). Mean OAR satisfaction score was 4.1 for CNS, 4.4 for H&N, and 4.6 for prostate structures. Overall CTV satisfaction score (n = 25), which encompassed the prostate, seminal vesicles, and neck lymph node volumes, was 4.1. CONCLUSIONS: Previously validated OAR DC models for CNS, H&N, and prostate RT planning required minimal subjective and objective edits and resulted in a positive user experience, although low survey compliance was a concern. CTV DC model evaluation was even more limited, but high user satisfaction suggests that they may have served as appropriate starting points for patient specific edits.

Monte Carlo simulations of EBT3 film dose deposition for percentage depth dose (PDD) curve evaluation.

PURPOSE: To use Monte Carlo (MC) calculations to evaluate the effects of Gafchromic EBT3 film orientation on percentage depth dose (PDD) curves. METHODS: Dose deposition in films placed in a water phantom, and oriented either parallel or perpendicular with respect to beam axis, were simulated with MC and compared to PDDs scored in a homogenous water phantom. The effects of introducing 0.01-1.00 mm air gaps on each side of the film as well as a small 1°-3° tilt for film placed in parallel orientation were studied. PDDs scored based on two published EBT3 film compositions were compared. Three photon beam energies of 120 kVp, 220 kVp, and 6 MV and three field sizes between 1 × 1 and 5 × 5 cm2 were considered. Experimental PDDs for a 6-MV 3 × 3 cm2 beam were acquired. RESULTS: PDD curves for films in perpendicular orientation more closely agreed to water PDDs than films placed in parallel orientation. The maximum difference between film and water PDD for films in parallel orientation was -12.9% for the 220 kVp beam. For the perpendicular film orientation, the maximum difference decreased to 5.7% for the 120 kVp beam. The inclusion of an air gap had the largest effect on the 6-MV 1 × 1 cm2 beam, for which the dose in the buildup region was underestimated by 21.2% compared to the simulation with no air gap. A 2° film tilt decreased the difference between the parallel film and homogeneous water phantom PDDs from -5.0% to -0.5% for the 6 MV 3 × 3 cm2 beam. The "newer" EBT3 film composition resulted in larger PDD discrepancies than the previous composition. Experimental film data qualitatively agreed with MC simulations. CONCLUSIONS: PDD measurements with films should either be performed with film in perpendicular orientation to the beam axis or in parallel orientation with a ~ 2º tilt and no air gaps.

Comparing deep learning-based auto-segmentation of organs at risk and clinical target volumes to expert inter-observer variability in radiotherapy planning.

BACKGROUND: Deep learning-based auto-segmented contours (DC) aim to alleviate labour intensive contouring of organs at risk (OAR) and clinical target volumes (CTV). Most previous DC validation studies have a limited number of expert observers for comparison and/or use a validation dataset related to the training dataset. We determine if DC models are comparable to Radiation Oncologist (RO) inter-observer variability on an independent dataset. METHODS: Expert contours (EC) were created by multiple ROs for central nervous system (CNS), head and neck (H&N), and prostate radiotherapy (RT) OARs and CTVs. DCs were generated using deep learning-based auto-segmentation software trained by a single RO on publicly available data. Contours were compared using Dice Similarity Coefficient (DSC) and 95% Hausdorff distance (HD). RESULTS: Sixty planning CT scans had 2-4 ECs, for a total of 60 CNS, 53 H&N, and 50 prostate RT contour sets. The mean DC and EC contouring times were 0.4 vs 7.7 min for CNS, 0.6 vs 26.6 min for H&N, and 0.4 vs 21.3 min for prostate RT contours. There were minimal differences in DSC and 95% HD involving DCs for OAR comparisons, but more noticeable differences for CTV comparisons. CONCLUSIONS: The accuracy of DCs trained by a single RO is comparable to expert inter-observer variability for the RT planning contours in this study. Use of deep learning-based auto-segmentation in clinical practice will likely lead to significant benefits to RT planning workflow and resources.

Synthesis of Heterologous Mevalonic Acid Pathway Enzymes in Clostridium ljungdahlii for the Conversion of Fructose and of Syngas to Mevalonate and Isoprene.

There is a growing interest in the use of microbial fermentation for the generation of high-demand, high-purity chemicals using cheap feedstocks in an environmentally friendly manner. One example explored here is the production of isoprene (C5H8), a hemiterpene, which is primarily polymerized to polyisoprene in synthetic rubber in tires but which can also be converted to C10 and C15 biofuels. The strictly anaerobic, acetogenic bacterium Clostridium ljungdahlii, used in all of the work described here, is capable of glycolysis using the Embden-Meyerhof-Parnas pathway and of carbon fixation using the Wood-Ljungdahl pathway. Clostridium-Escherichia coli shuttle plasmids, each bearing either 2 or 3 different heterologous genes of the eukaryotic mevalonic acid (MVA) pathway or eukaryotic isopentenyl pyrophosphate isomerase (Idi) and isoprene synthase (IspS), were constructed and electroporated into C. ljungdahlii These plasmids, one or two of which were introduced into the host cells, enabled the synthesis of mevalonate and of isoprene from fructose and from syngas (H2, CO2, and CO) and the conversion of mevalonate to isoprene. All of the heterologous enzymes of the MVA pathway, as well as Idi and IspS, were shown to be synthesized at high levels in C. ljungdahlii, as demonstrated by Western blotting, and were enzymatically active, as demonstrated by in vivo product synthesis. The quantities of mevalonate and isoprene produced here are far below what would be required of a commercial production strain. However, proposals are made that could enable a substantial increase in the mass yield of product formation.IMPORTANCE This study demonstrates the ability to synthesize a heterologous metabolic pathway in C. ljungdahlii, an organism capable of metabolizing either simple sugars or syngas or both together (mixotrophy). Syngas, an inexpensive source of carbon and reducing equivalents, is produced as a major component of some industrial waste gas, and it can be generated by gasification of cellulosic biowaste and of municipal solid waste. Its conversion to useful products therefore offers potential cost and environmental benefits. The ability of C. ljungdahlii to grow mixotrophically also enables the recapture, should there be sufficient reducing equivalents available, of the CO2 released upon glycolysis, potentially increasing the mass yield of product formation. Isoprene is the simplest of the terpenoids, and so the demonstration of its production is a first step toward the synthesis of higher-value products of the terpenoid pathway.

Radiation-induced refraction artifacts in the optical CT readout of polymer gel dosimeters.

PURPOSE: The objective of this work is to demonstrate imaging artifacts that can occur during the optical computed tomography (CT) scanning of polymer gel dosimeters due to radiation-induced refractive index (RI) changes in polyacrylamide gels. METHODS: A 1 L cylindrical polyacrylamide gel dosimeter was irradiated with 3 × 3 cm(2) square beams of 6 MV photons. A prototype fan-beam optical CT scanner was used to image the dosimeter. Investigative optical CT scans were performed to examine two types of rayline bending: (i) bending within the plane of the fan-beam and (ii) bending out the plane of the fan-beam. To address structured errors, an iterative Savitzky-Golay (ISG) filtering routine was designed to filter 2D projections in sinogram space. For comparison, 2D projections were alternatively filtered using an adaptive-mean (AM) filter. RESULTS: In-plane rayline bending was most notably observed in optical CT projections where rays of the fan-beam confronted a sustained dose gradient that was perpendicular to their trajectory but within the fan-beam plane. These errors caused distinct streaking artifacts in image reconstructions due to the refraction of higher intensity rays toward more opaque regions of the dosimeter. Out-of-plane rayline bending was observed in slices of the dosimeter that featured dose gradients perpendicular to the plane of the fan-beam. These errors caused widespread, severe overestimations of dose in image reconstructions due to the higher-than-actual opacity that is perceived by the scanner when light is bent off of the detector array. The ISG filtering routine outperformed AM filtering for both in-plane and out-of-plane rayline errors caused by radiation-induced RI changes. For in-plane rayline errors, streaks in an irradiated region (>7 Gy) were as high as 49% for unfiltered data, 14% for AM, and 6% for ISG. For out-of-plane rayline errors, overestimations of dose in a low-dose region (∼50 cGy) were as high as 13 Gy for unfiltered data, 10 Gy for AM, and 3.1 Gy for ISG. The ISG routine also addressed unrelated artifacts that previously needed to be manually removed in sinogram space. However, the ISG routine blurred reconstructions, causing losses in spatial resolution of ∼5 mm in the plane of the fan-beam and ∼8 mm perpendicular to the fan-beam. CONCLUSIONS: This paper reveals a new category of imaging artifacts that can affect the optical CT readout of polyacrylamide gel dosimeters. Investigative scans show that radiation-induced RI changes can cause significant rayline errors when rays confront a prolonged dose gradient that runs perpendicular to their trajectory. In fan-beam optical CT, these errors manifested in two ways: (1) distinct streaking artifacts caused by in-plane rayline bending and (2) severe overestimations of opacity caused by rays bending out of the fan-beam plane and missing the detector array. Although the ISG filtering routine mitigated these errors better than an adaptive-mean filtering routine, it caused unacceptable losses in spatial resolution.

A prototype fan-beam optical CT scanner for 3D dosimetry.

PURPOSE: The objective of this work is to introduce a prototype fan-beam optical computed tomography scanner for three-dimensional (3D) radiation dosimetry. METHODS: Two techniques of fan-beam creation were evaluated: a helium-neon laser (HeNe, λ = 543 nm) with line-generating lens, and a laser diode module (LDM, λ = 635 nm) with line-creating head module. Two physical collimator designs were assessed: a single-slot collimator and a multihole collimator. Optimal collimator depth was determined by observing the signal of a single photodiode with varying collimator depths. A method of extending the dynamic range of the system is presented. Two sample types were used for evaluations: nondosimetric absorbent solutions and irradiated polymer gel dosimeters, each housed in 1 liter cylindrical plastic flasks. Imaging protocol investigations were performed to address ring artefacts and image noise. Two image artefact removal techniques were performed in sinogram space. Collimator efficacy was evaluated by imaging highly opaque samples of scatter-based and absorption-based solutions. A noise-based flask registration technique was developed. Two protocols for gel manufacture were examined. RESULTS: The LDM proved advantageous over the HeNe laser due to its reduced noise. Also, the LDM uses a wavelength more suitable for the PRESAGE(TM) dosimeter. Collimator depth of 1.5 cm was found to be an optimal balance between scatter rejection, signal strength, and manufacture ease. The multihole collimator is capable of maintaining accurate scatter-rejection to high levels of opacity with scatter-based solutions (T < 0.015%). Imaging protocol investigations support the need for preirradiation and postirradiation scanning to reduce reflection-based ring artefacts and to accommodate flask imperfections and gel inhomogeneities. Artefact removal techniques in sinogram space eliminate streaking artefacts and reduce ring artefacts of up to ∼40% in magnitude. The flask registration technique was shown to achieve submillimetre and subdegree placement accuracy. Dosimetry protocol investigations emphasize the need to allow gel dosimeters to cool gradually and to be scanned while at room temperature. Preliminary tests show that considerable noise reduction can be achieved with sinogram filtering and by binning image pixels into more clinically relevant grid sizes. CONCLUSIONS: This paper describes a new optical CT scanner for 3D radiation dosimetry. Tests demonstrate that it is capable of imaging both absorption-based and scatter-based samples of high opacities. Imaging protocol and gel dosimeter manufacture techniques have been adapted to produce optimal reconstruction results. These optimal results will require suitable filtering and binning techniques for noise reduction purposes.

CT, MR, and ultrasound image artifacts from prostate brachytherapy seed implants: the impact of seed size.

PURPOSE: To investigate the effects of brachytherapy seed size on the quality of x-ray computed tomography (CT), ultrasound (US), and magnetic resonance (MR) images and seed localization through comparison of the 6711 and 9011 (125)I sources. METHODS: For CT images, an acrylic phantom mimicking a clinical implantation plan and embedded with low contrast regions of interest (ROIs) was designed for both the 0.774 mm diameter 6711 (standard) and the 0.508 mm diameter 9011 (thin) seed models (Oncura, Inc., and GE Healthcare, Arlington Heights, IL). Image quality metrics were assessed using the standard deviation of ROIs between the seeds and the contrast to noise ratio (CNR) within the low contrast ROIs. For US images, water phantoms with both single and multiseed arrangements were constructed for both seed sizes. For MR images, both seeds were implanted into a porcine gel and imaged with pelvic imaging protocols. The standard deviation of ROIs and CNR values were used as metrics of artifact quantification. Seed localization within the CT images was assessed using the automated seed finder in a commercial brachytherapy treatment planning system. The number of erroneous seed placements and the average and maximum error in seed placements were recorded as metrics of the localization accuracy. RESULTS: With the thin seeds, CT image noise was reduced from 48.5 ± 0.2 to 32.0 ± 0.2 HU and CNR improved by a median value of 74% when compared with the standard seeds. Ultrasound image noise was measured at 50.3 ± 17.1 dB for the thin seed images and 50.0 ± 19.8 dB for the standard seed images, and artifacts directly behind the seeds were smaller and less prominent with the thin seed model. For MR images, CNR of the standard seeds reduced on average 17% when using the thin seeds for all different imaging sequences and seed orientations, but these differences are not appreciable. Automated seed localization required an average (±SD) of 7.0 ± 3.5 manual corrections in seed positions for the thin seed scans and 3.0 ± 1.2 manual corrections in seed positions for the standard seed scans. The average error in seed placement was 1.2 mm for both seed types and the maximum error in seed placement was 2.1 mm for the thin seed scans and 1.8 mm for the standard seed scans. CONCLUSIONS: The 9011 thin seeds yielded significantly improved image quality for CT and US images but no significant differences in MR image quality.

Hemin binding protein C is found in outer membrane vesicles and protects Bartonella henselae against toxic concentrations of hemin.

Bartonella species are gram-negative, emerging bacterial pathogens found in two distinct environments. In the gut of the obligately hematophagous arthropod vector, bartonellae are exposed to concentrations of heme that are toxic to other bacteria. In the bloodstream of the mammalian host, access to heme and iron is severely restricted. Bartonellae have unusually high requirements for heme, which is their only utilizable source of iron. Although heme is essential for Bartonella survival, little is known about genes involved in heme acquisition and detoxification. We developed a strategy for high-efficiency transposon mutagenesis to screen for genes in B. henselae heme binding and uptake pathways. We identified a B. henselae transposon mutant that constitutively expresses the hemin binding protein C (hbpC) gene. In the wild-type strain, transcription of B. henselae hbpC was upregulated at arthropod temperature (28°C), compared to mammalian temperature (37°C). In the mutant strain, temperature-dependent regulation was absent. We demonstrated that HbpC binds hemin and localizes to the B. henselae outer membrane and outer membrane vesicles. Overexpression of hbpC in B. henselae increased resistance to heme toxicity, implicating HbpC in protection of B. henselae from the toxic levels of heme present in the gut of the arthropod vector. Experimental inoculation of cats with B. henselae strains demonstrated that both constitutive expression and deletion of hbpC affect the ability of B. henselae to infect the cat host. Modulation of hbpC expression appears to be a strategy employed by B. henselae to survive in the arthropod vector and the mammalian host.

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei.

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.

CT image artifacts from brachytherapy seed implants: a postprocessing 3D adaptive median filter.

PURPOSE: To design a postprocessing 3D adaptive median filter that minimizes streak artifacts and improves soft-tissue contrast in postoperative CT images of brachytherapy seed implantations. METHODS: The filter works by identifying voxels that are likely streaks and estimating more reflective voxel intensity by using voxel intensities in adjacent CT slices and applying a median filter over voxels not identified as seeds. Median values are computed over a 5 x 5 x 5 mm region of interest (ROI) within the CT volume. An acrylic phantom simulating a clinical seed implant arrangement and containing nonradioactive seeds was created. Low contrast subvolumes of tissuelike material were also embedded in the phantom. Pre- and postprocessed image quality metrics were compared using the standard deviation of ROIs between the seeds, the CT numbers of low contrast ROIs embedded within the phantom, the signal to noise ratio (SNR), and the contrast to noise ratio (CNR) of the low contrast ROIs. The method was demonstrated with a clinical postimplant CT dataset. RESULTS: After the filter was applied, the standard deviation of CT values in streak artifact regions was significantly reduced from 76.5 to 7.2 HU. Within the observable low contrast plugs, the mean of all ROI standard deviations was significantly reduced from 60.5 to 3.9 HU, SNR significantly increased from 2.3 to 22.4, and CNR significantly increased from 0.2 to 4.1 (all P < 0.01). The mean CT in the low contrast plugs remained within 5 HU of the original values. CONCLUSION: An efficient postprocessing filter that does not require access to projection data, which can be applied irrespective of CT scan parameters has been developed, provided the slice thickness and spacing is 3 mm or less.

Respiratory Gating: Using Deep Inspiration Breath Hold Radiation Therapy to Treat Left Breast Cancer.

Radiation therapy with respiratory gating has typically been used in the treatment of lung tumours. This technique offers clinical advantages when applied to other sites as well. This is a case report of a 47-year-old woman who presented with a localized recurrent breast cancer in the left 6th intercostal space and declined a chest wall resection. Standard radiation therapy carried a significant risk of cardiac toxicity. A respiratory gated, deep inspiration breath-hold technique (DIBH) was developed that significantly reduced the dose to her heart and ipsilateral lung while delivering a curative dose (70 Gy) to the recurrent tumour. Treatment accuracy, setup, reproducibility, and daily treatment times were measured. DIBH was tolerable, reproducible, and, after the first week, could be delivered within a 15-minute treatment slot. Toxicity was minimal.

Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide.

The enteric pathogen Campylobacter jejuni is a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence in C. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found that C. jejuni reacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring beta1-3 and/or beta1-4 linkages. CFW reactivity increased with extended growth, under 42 degrees C anaerobic conditions, and in a DeltaspoT mutant defective for the stringent response (SR). Conversely, two newly isolated dim mutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and DeltaspoT and dim mutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, and N-linked polysaccharides. Targeted deletion of carB downstream of the dim13 mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with the C. jejuni DeltaspoT mutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production in C. jejuni.

ExoR is genetically coupled to the ExoS-ChvI two-component system and located in the periplasm of Sinorhizobium meliloti.

Sinorhizobium meliloti enters into a symbiotic relationship with legume host plants, providing fixed nitrogen in exchange for carbon and amino acids. In S. meliloti, exoR and the exoS-chvI two-component system regulate the biosynthesis of succinoglycan, an exopolysaccharide important for host invasion. It was previously reported that a loss-of-function mutation in exoR and a gain-of-function mutation in exoS cause overproduction of succinoglycan and loss of motility, indicating that ExoR negatively regulates and ExoS-ChvI positively regulates downstream genes. However, a relationship between exoR and exoS-chvI has never been clearly established. By identification and detailed characterization of suppressor strains, we provide genetic evidence that exoR and exoS-chvI control many similar phenotypes. These include succinoglycan production, symbiosis, motility, and previously uncharacterized prototrophy and biofilm formation, all of which are co-ordinately restored by suppressors. We further demonstrate that ExoR is located in the periplasm, suggesting that it functions to regulate downstream genes in a novel manner. In pathogenic bacteria closely related to S. meliloti, exoS-chvI homologues are required for virulence and the regulation of cell envelope composition. Our data suggest that periplasmically localized ExoR and ExoS-ChvI function together in a unique and critical regulatory system associated with both free-living and symbiotic states of S. meliloti.

Helicobacter pylori initiates the stringent response upon nutrient and pH downshift.

Helicobacter pylori was previously reported to lack a stringent response. In contrast, we show that after nutrient downshift, H. pylori produced abundant ppGpp and less total RNA. pH downshift also caused (p)ppGpp accumulation. Our observations indicate that nutrient deprivation and acid shock activate the stringent response in H. pylori.

The Campylobacter jejuni stringent response controls specific stress survival and virulence-associated phenotypes.

Campylobacter jejuni is a highly prevalent food-borne pathogen that causes diarrhoeal disease in humans. A natural zoonotic, it must overcome significant stresses both in vivo and during transmission despite the absence of several traditional stress response genes. Although relatively little is understood about its mechanisms of pathogenesis, its ability to interact with and invade human intestinal epithelial cells closely correlates with virulence. A C. jejuni microarray-based screen revealed that several known virulence genes and several uncharacterized genes, including spoT, were rapidly upregulated during infection of human epithelial cells. spoT and its homologue relA have been shown in other bacteria to regulate the stringent response, an important stress response that to date had not been demonstrated for C. jejuni or any other epsilon-proteobacteria. We have found that C. jejuni mounts a stringent response that is regulated by spoT. Detailed analyses of a C. jejuni delta spoT mutant revealed that the stringent response is required for several specific stress, transmission and antibiotic resistance-related phenotypes. These include stationary phase survival, growth and survival under low CO2/high O2 conditions, and rifampicin resistance. A secondary suppressor strain that specifically rescues the low CO2 growth defect of the delta spoT mutant was also isolated. The stringent response additionally proved to be required for the virulence-related phenotypes of adherence, invasion, and intracellular survival in two human epithelial cell culture models of infection; spoT is the first C. jejuni gene shown to participate in longer term survival in epithelial cells. Microarray analyses comparing wild-type to the delta spoT mutant also revealed a strong correlation between gene expression profiles and phenotype differences observed. Together, these data demonstrate a critical role for the C. jejuni stringent response in multiple aspects of C. jejuni biology and pathogenesis and, further, may lend novel insight into unexplored features of the stringent response in other prokaryotic organisms.

Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.

The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

Electron energy constancy verification using a double-wedge phantom.

Routine constancy checks of electron energy are often time consuming because of the necessity to measure a dose at two depths. A technique is described that uses a double-wedge shaped phantom positioned on a Profiler diode array for measuring an electron energy constancy metric similar to R(50). The double-wedge electron profiles are invariant to phantom alignment in the wedge direction, unlike single wedge techniques, and the sensitivity of the technique is similar to water-based depth-dose measurements over an energy range of 6 to 20 MeV. Reproducibility results ranging from 0.01 to 0.03 cm were achieved for measurements taken over the course of 1.5 yrs. The technique is efficient in that only one phantom setup is required for all electron energies.

Analysis of differences between Sinorhizobium meliloti 1021 and 2011 strains using the host calcium spiking response.

In the Rhizobium-legume symbiosis, compatible partners recognize each other through an exchange of signals. Plant inducers act together with bacterial transcriptional activators, the NodD proteins, to regulate the expression of bacterial biosynthetic nodulation (nod) genes. These genes direct the synthesis of a lipochito-oligosaccharide signal called Nod factor (NF). NFs elicit an early host response, root hair calcium spiking, that is initiated in root hair cells within 15 min of NF or live Rhizobium inoculation. We used calcium spiking as an assay to compare two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011, derived from the same field isolate. We found that the two strains show a kinetic difference in the calcium spiking assay: Rm1021 elicits calcium spiking in host root hairs as rapidly as purified NF, whereas Rm2011 shows a significant delay. This difference can be overcome by raising expression levels of either the NodD transcriptional activators or GroEL, a molecular chaperone that affects expression of the biosynthetic nod genes. We further demonstrate that the delay in triggering calcium spiking exhibited by Rm2011 is correlated with a reduced amount of nod gene expression compared with Rm1021. Therefore, calcium spiking is a useful tool in detecting subtle differences in bacterial gene expression that affect the early stages of the Rhizobium-legume symbiosis.

The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis.

Sinorhizobium meliloti and host legumes enter into a nitrogen-fixing, symbiotic relationship triggered by an exchange of signals between bacteria and plant. S. meliloti produces Nod factor, which elicits the formation of nodules on plant roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion and colonization of the host. The biosynthesis of these molecules is well defined, but the specific regulation of these compounds is not completely understood. Bacteria control complex regulatory networks by the production of ppGpp, the effector molecule of the stringent response, which induces physiological change in response to adverse growth conditions and can also control bacterial development and virulence. Through detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we show that the stringent response is required for nodule formation and regulates the production of succinoglycan. Although it remains unknown whether these phenotypes are connected, we have isolated suppressor strains that restore both defects and potentially identify key downstream regulatory genes. These results indicate that the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.