Enhanced transport of bacteria along root systems by protists can impact plant health.

Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpoda sp.), a flagellate (Cercomonas sp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixing Sinorhizobium meliloti to infectible root tips. Co-inoculation of protists plus S. meliloti resulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated with S. meliloti alone. Co-inoculation of the ciliate, Colpoda sp., with S. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix. Colpoda sp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum of S. meliloti resulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling.IMPORTANCEProtists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria, Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S. meliloti alone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.

Soil Protists Can Actively Redistribute Beneficial Bacteria along Medicago truncatula Roots.

The rhizosphere is the region of soil directly influenced by plant roots. The microbial community in the rhizosphere includes fungi, protists, and bacteria: all play significant roles in plant health. The beneficial bacterium Sinorhizobium meliloti infects growing root hairs on nitrogen-starved leguminous plants. Infection leads to the formation of a root nodule, where S. meliloti converts atmospheric nitrogen to ammonia, a bioavailable form. In soil, S. meliloti is often found in biofilms and travels slowly along the roots, leaving developing root hairs at the growing root tips uninfected. Soil protists are an important component of the rhizosphere system, able to travel quickly along roots and water films, who prey on soil bacteria and have been known to egest undigested phagosomes. We show that a soil protist, Colpoda sp., can transport S. meliloti down Medicago truncatula roots. Using model soil microcosms, we directly observed fluorescently labeled S. meliloti along M. truncatula roots and tracked the displacement of the fluorescence signal over time. Two weeks after co-inoculation, this signal extended 52 mm farther down plant roots when Colpoda sp. was also present versus treatments that contained bacteria but not protists. Direct counts also showed protists are required for viable bacteria to reach the deeper sections of our microcosms. Facilitating bacterial transport may be an important mechanism whereby soil protists promote plant health. IMPORTANCE Soil protists are an important part of the microbial community in the rhizosphere. Plants grown with protists fare better than plants grown without protists. Mechanisms through which protists support plant health include nutrient cycling, alteration of the bacterial community through selective feeding, and consumption of plant pathogens. Here, we provide data in support of an additional mechanism: protists act as transport vehicles for bacteria in soil. We show that protist-facilitated transport can deliver plant-beneficial bacteria to the growing tips of roots that may otherwise be sparsely inhabited with bacteria originating from a seed-associated inoculum. By co-inoculating Medicago truncatula roots with both S. meliloti, a nitrogen-fixing legume symbiont, and Colpoda sp., a ciliated protist, we show substantial and statistically significant transport with depth and breadth of bacteria-associated fluorescence as well as transport of viable bacteria. Co-inoculation with shelf-stable encysted soil protists may be employed as a sustainable agriculture biotechnology to better distribute beneficial bacteria and enhance the performance of inoculants.

Causes of infant deaths and patterns of associated factors in Eastern Ethiopia: Results of verbal autopsy (InterVA-4) study.

BACKGROUND: In a range of setting, detecting and generate empirical information on the cause of infant death and contributing risk factors at population level is basically utmost essential to take evidence-based measures in reducing infant morbidity and mortality. An electronic verbal autopsy is suitable tool and best alternative solution to determine individuals' cause of death in a setting where the majority of deaths occur at home and civil registration systems do not exist. The present study was undertaken to find out cause of infant death, applying computer-based probabilistic model (InterVA-4) and analyze the patterns of association factors of mother's and the deceased infant's characteristics to the leading cause-specific infant mortality in Eastern Ethiopia. METHODS: The study employed a community-based prospective longitudinal survey, which was conducted with routinely enumeration of reported infant deaths for a period of two years (from September 2016 to August 2018) in Eastern part of Ethiopia. Using the two-stage cluster sampling technique, the study was undertaken in four randomly selected districts of West Hararghe zone and two districts of zone 3 in Oromia and Afar regional state, respectively. The study included a total of 362 infants who were deceased during the study period. Data was collected by trained enumerators by interviewing the mothers or guardians of the deceased infant using a 2014 standardize World Health Organization (WHO) Verbal Autopsy questionnaire. InterVA-4 model were used for processing and interpreting verbal autopsy data in order to arrive at the most likely causes of infant death. SPSS version 23 was also used for statistical analysis of frequency distribution and logistic regression for the association between covariates and outcomes. FINDINGS: Of the overall (362) deceased infants' during the study period, 53.0% of deaths occurred during neonatal time while 47.0% died in the post-neonatal period. Acute respiratory infection including neonatal and post-neonatal pneumonia (38.4%), birth asphyxia (16.4%), diarrheal diseases (16.3%), prematurity (7.4%) and malaria (4.3%) were found to be the leading causes of infant mortality in the study area. The independent factors strongly associated with probable ARI, including pneumonia related mortality as compared to all-causes of death were infants with maternal age lower than 20 years old (p = 0.001, AOR: 4.82, 95% CI: 1.88, 12.3) and infant being died outside of heath facilities (P = 0.007, AOR: 2.85, 95% CI: 1.33, 6.12). The post-neonatal period (P = 0.000, AOR: 15.5, 95% CI: 6.35, 37.8) and infant died in the wet season (P = 0.006, AOR: 2.38, 95% CI: 1.28, 4.44) had strong relationship with dying from diarrhea-related death than those infants died from all non-diarrhea. The death due to malaria robustly associated with infants whose mothers age between 20-35 years old (P = 0.024, AOR: 4.44, 95% CI: 1.22, 16.2) and infant who was dwelled in the districts of Afar region (P = 0.013, AOR: 4.08, 95% CI: 1.35, 12.4). CONCLUSION: The highest cause of infant mortality was associated with disease of respiratory system, particularly acute respiratory infection, including both neonates and post-neonatal pneumonia. Most of the infant deaths existed are as a result of diseases and conditions that are readily preventable or treatable cause, similar to those reported in worldwide, which have needs of further attention. The patterns of significant associated factors across cause-specific mortality against all-cause of death were dissimilar. Therefore, strengthen maternal and child health program with effective preventive interventions emphasizing on the most common cause of infant deaths and those factors contributing in raising mortality risk are required.

The risk of water, sanitation and hygiene on diarrhea-related infant mortality in eastern Ethiopia: a population-based nested case-control.

BACKGROUND: Diarrhea is still appeared to be as one of the leading global killers and disability-adjusted life-years lost, particularly in the infant and children. As per WHO, about 88% of diarrhea-related deaths are attributable to unsafe water, inadequate sanitation and insufficient hygiene, mainly in developing world. Thus, the main objective of this study was to find out the risk of such factors that contribute for diarrhea-related infant mortality in Eastern Ethiopia. METHODS: This study employed community based unmatched nested case-control study design in Eastern Ethiopia. The cases were infants who died from diarrheal disease while controls were those who survived their first year of life from September, 2016 to August, 2018. A total of 305 study subjects (61 cases and 244 controls) were included in the study. Infants dying from diarrhea were compared to four neighborhood controls in terms of several risk components of Water, Sanitation and Hygiene. Data were collected from mothers/care takers of infants using pre-tested structured questionnaires, and entered onto CSpro version 5.1 and transform to SPSS version 23 to analyzed potential risk factors. FINDINGS: Finding of this study revealed that the risk factors that found to be significantly associated with infant death from diarrhoea after adjustment for confounding variables included the age of mother with < 20 years old (P = 0.009, AOR: 0.01, 95% CI: 0.01, 0.47), unsafe drinking water storage (P = 0.013, AOR: 0.4, 95% CI: 0.18, 0.81), infants in households without point-of-use water treatment practices (P = 0.004, AOR: 0.21, 95% CI: 0.08, 0.61), households with unimproved sanitation (P = 0.050, AOR: 0.36, 95% CI: 0.13, 1.00), unsafe disposing of child feces (P = 0.014, AOR: 0.34, 95% CI: 0.15, 0.81), and improper management of solid waste (P = 0.003, AOR: 0.29, 95% CI: 0.13, 0.66). These exposure factors had lower risk for the contribution of infants dying from diarrhoea than those with their reference group in the study area. However, infants in households with improper management of liquid waste management showed strongly significant association which had three times more likely to occur diarrhea-related infant death (P = 0.010, AOR: 3.43, 95% CI: 1.34, 8.76). Similarly, infants whose mother/caretaker practiced hand washing with less critical time (one-two occasions) had three times greater risk to infant death from diarrhea than those who had practice more than three critical times of hand washing (P = 0.027, AOR: 3.04, 95% CI: 1.13, 8.17). CONCLUSION: This study suggests that infants in households with improper management of liquid waste and hand washing practices with fewer occasions (one-two critical time) are a greater risk of getting a diarrhea-related infant death. Therefore, efforts should be made to ensure intervention taking such risk factors into consideration, typically in the infantile period.

Simultaneous Single-Cell Genome and Transcriptome Sequencing of Termite Hindgut Protists Reveals Metabolic and Evolutionary Traits of Their Endosymbionts.

Some of the protist species which colonize the hindguts of wood-feeding Reticulitermes termites are associated with endosymbiotic bacteria belonging to the genus Endomicrobium. In this study, we focused on the endosymbionts of three protist species from Reticulitermes flavipes, as follows: Pyrsonympha vertens, Trichonympha agilis, and Dinenympha species II. Since these protist hosts represented members of different taxa which colonize separate niches within the hindguts of their termite hosts, we investigated if these differences translated to differential gene content and expression in their endosymbionts. Following assembly and comparative genome and transcriptome analyses, we discovered that these endosymbionts differed with respect to some possible niche-specific traits, such as carbon metabolism. Our analyses suggest that species-specific genes related to carbon metabolism were acquired by horizontal gene transfer (HGT) and may have come from taxa which are common in the termite hind gut. In addition, our analyses suggested that these endosymbionts contain and express genes related to natural transformation (competence) and recombination. Taken together, the presence of genes acquired by HGT and a putative competence pathway suggest that these endosymbionts are not cut off from gene flow and that competence may be a mechanism by which members of Endomicrobium can acquire new traits. IMPORTANCE The composition and structure of wood, which contains cellulose, hemicellulose, and lignin, prevent most organisms from using this common food source. Termites are a rare exception among animals, and they rely on a complex microbiota housed in their hindguts to use wood as a source of food. The lower termite, Reticulitermes flavipes, houses a variety of protists and prokaryotes that are the key players in the disassembly of lignocellulose. Here, we describe the genomes and the gene expression profiles of five Endomicrobium endosymbionts living inside three different protist species from R. flavipes. Data from these genomes suggest that these Endomicrobium species have different mechanisms for using carbon. In addition, they harbor genes that may be used to import DNA from their environment. This process of DNA uptake may contribute to the high levels of horizontal gene transfer noted previously in Endomicrobium species.

18S rRNA gene amplicon sequencing combined with culture-based surveys of maize rhizosphere protists reveal dominant, plant-enriched and culturable community members.

Protists play important roles in shaping the microbial community of the rhizosphere and defining these roles will require the study of protist isolates. However, there is still a limited understanding of how well protist isolation efforts can capture the diversity and composition of rhizosphere protistan communities. Here, we report a simultaneous isolation and 18S rRNA gene amplicon sequencing survey describing the protist diversity of maize rhizospheres in two climatically and pedologically distinct sites. We demonstrated that the maize rhizosphere exerted significant and site-dependent effects on the protistan community structure and defined a set of core and rhizosphere-enriched protists. From the same root samples, we generated a library of 103 protist isolates representing 46 18S rRNA gene sequence variants from six eukaryotic supergroups. While cultured isolates represented a small proportion of total protist diversity recovered by sequencing, they included taxa enriched in rhizosphere soils across all samples, encompassing 9% of all core sequence variants. The isolation approach also captured 17 protists not detected through 18S rRNA gene amplicon sequencing. This study demonstrated that maize roots select for distinct protistan communities, and established a diverse protist culture collection that can be used for future research linking protists to rhizosphere status and plant health.

Draft Genome Sequences of Dysgonomonas sp. Strains GY75 and GY617, Isolated from the Hindgut of Reticulitermes flavipes.

Dysgonomonas species are facultative heterotrophs capable of growth on lignocellulose-derived polysaccharides. Dysgonomonas species harbor myriad genes involved in glycan modification and are well suited to the lignocellulose-rich conditions within the termite hindgut. Here, we report draft genome sequences for Dysgonomonas sp. strains GY75 and GY617, isolated from the hindgut of Reticulitermes flavipes.

Draft Genome Sequences of Dysgonomonas sp. Strains BGC7 and HGC4, Isolated from the Hindgut of a Lower Termite.

Dysgonomonas spp. are facultative heterotrophs which colonize diverse environments, including the hindgut of the lower termite Reticulitermes flavipes Dysgonomonas genomes are enriched for genes involving oligo- and polysaccharide utilization, enabling modification of a wide array of complex glycans. Here, we report draft genome sequences for Dysgonomonas sp. strains BGC7 and HGC4.

Optogenetics in Sinorhizobium meliloti Enables Spatial Control of Exopolysaccharide Production and Biofilm Structure.

Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.

Development and application of aerobic, chemically defined media for Dysgonomonas.

Members of Dysgonomonas are Gram-stain-negative, non-motile, facultatively anaerobic coccobacilli originally described in relation to their isolation from stool and wounds of human patients (CDC group DF-3). More recently, Dysgonomonas have been found to be widely distributed in terrestrial environments and are particularly enriched in insect systems. Their prevalence in xylophagous insects such as termites and wood-feeding cockroaches, as well as in soil-fed microbial fuel cells, elicit interest in lignocellulose degradation and biofuel production, respectively. Their occurrence in mosquito and fruit fly have implications relating to symbiosis, host immunology and developmental biology. Additionally, their presence in termite, mosquito and nematode present novel opportunities for pest and vector control. Currently, the absolute growth requirements of Dysgonomonas are unknown, and they are commonly cultured under anaerobic conditions on complex media containing blood, peptones, tryptones, and yeast, plant or meat extracts. Restrictive and undefined culturing conditions preclude physiological and genetic studies, and thus further understanding of their metabolic potential. Here we describe the requirements for growth of termite-derived Dysgonomonas isolates and create parallel complex, defined and minimal media that permit vigorous and reliable aerobic growth. Furthermore, we show that these media can be used to easily enrich for Dysgonomonas isolates from densely-colonized and microbially-diverse environmental samples.

Single-cell amplicon sequencing reveals community structures and transmission trends of protist-associated bacteria in a termite host.

The hindgut protists of wood-feeding termites are usually colonized by prokaryotic symbionts. Many of the hurdles that have prevented a better understanding of these symbionts arise from variation among protist and termite host species and the inability to maintain prominent community members in culture. These issues have made it difficult to study the fidelity, acquisition, and differences in colonization of protists by bacterial symbionts. In this study, we use high throughput amplicon sequencing of the V4 region of 16S rRNA genes to determine the composition of bacterial communities associated with single protist cells of six protist species, from the genera Pyrsonympha, Dinenympha, and Trichonympha that are present in the hindgut of the termite Reticulitermes flavipes. By analyzing amplicon sequence variants (ASVs), the diversity and distribution of protist-associated bacteria was compared within and across these six different protist species. ASV analysis showed that, in general, each protist genus associated with a distinct community of bacterial symbionts which were conserved across different termite colonies. However, some ASVs corresponding to ectosymbionts (Spirochaetes) were shared between different Dinenympha species and to a lesser extent with Pyrsonympha and Trichonympha hosts. This suggested that certain bacterial symbionts may be cosmopolitan to some degree and perhaps acquired by horizontal transmission. Using a fluorescence-based cell assay, we could observe the horizontal acquisition of surface-bound bacteria. This acquisition was shown to be time-dependent, involve active processes, and was non-random with respect to binding locations on some protists.

Protist-facilitated particle transport using emulated soil micromodels.

Microbial processes in the subsurface can be visualized directly using micromodels to emulate pore-scale geometries. Here, emulated soil micromodels were used to measure transport of fluorescent beads in the presence and absence of the soil ciliate Colpoda sp. under quiescent conditions. Beads alone or beads with protists were delivered to the input wells of replicate micromodels that contained three 20 mm(2) channels emulating a sandy loam microstructure. Bead abundance in microstructured channels was measured by direct counts of tiled confocal micrographs. For channels with protists, average bead abundances were approximately 320, 560, 710, 830, and 790 mm(-2) after 1, 2, 3, 5, and 10 days, respectively, versus 0, 0, 0.3, 7.8, and 45 mm(-2) without protists. Spatial and temporal patterns of bead abundance indicate that protist-facilitated transport is not a diffusive-type process but rather a function of more complex protist behaviors, including particle uptake and egestion and motility in a microstructured habitat. Protist-facilitated transport may enhance particle mixing in the soil subsurface and could someday be used for targeted delivery of nanoparticles, encapsulated chemicals, or bacteria for remediation and agriculture applications.

Biochemical characterization of a nitrogen-type phosphotransferase system reveals that enzyme EI(Ntr) integrates carbon and nitrogen signaling in Sinorhizobium meliloti.

In Sinorhizobium meliloti, catabolite repression is influenced by a noncanonical nitrogen-type phosphotransferase system (PTS(Ntr)). In this PTS(Ntr), the protein HPr is phosphorylated on histidine-22 by the enzyme EI(Ntr) and the flux of phosphate through this residue onto downstream proteins leads to an increase in succinate-mediated catabolite repression (SMCR). In order to explore the molecular determinants of HPr phosphorylation by EI(Ntr), both proteins were purified and the activity of EI(Ntr) was measured. Experimentally determined kinetic parameters of EI(Ntr) activity were significantly slower than those determined for the carbohydrate-type EI in Escherichia coli. Enzymatic assays showed that glutamine, a signal of nitrogen availability in many Gram-negative bacteria, strongly inhibits EI(Ntr). Binding experiments using the isolated GAF domain of EI(Ntr) (EIGAF) showed that it is the domain responsible for detection of glutamine. EI(Ntr) activity was not affected by α-ketoglutarate, and no binding between the EIGAF and α-ketoglutarate could be detected. These data suggest that in S. melilloti, EI(Ntr) phosphorylation of HPr is regulated by signals from both carbon metabolism (phosphoenolpyruvate) and nitrogen metabolism (glutamine).

NMR structure of the HWE kinase associated response regulator Sma0114 in its activated state.

Bacterial receiver domains modulate intracellular responses to external stimuli in two-component systems. Sma0114 is the first structurally characterized representative from the family of receiver domains that are substrates for histidine-tryptophan-glutamate (HWE) kinases. We report the NMR structure of Sma0114 bound by Ca(2+) and BeF3(-), a phosphate analogue that stabilizes the activated state. Differences between the NMR structures of the inactive and activated states occur in helix α1, the active site loop that connects strand ß3 and helix α3, and in the segment from strand ß5 to helix α5 of the 455 (α4-ß5-α5) face. Structural rearrangements of the 455 face typically make receiver domains competent for binding downstream target molecules. In Sma0114 the structural changes accompanying activation result in a more negatively charged surface for the 455 face. Coupling between the 455 face and active site phosphorylation is usually mediated through the rearrangement of a threonine and tyrosine residue, in a mechanism called Y-T coupling. The NMR structure indicates that Sma0114 lacks Y-T coupling and that communication between the active site and the 455 face is achieved through a conserved lysine residue that stabilizes the acyl phosphate in receiver domains. (15)N-NMR relaxation experiments were used to investigate the backbone dynamics of the Sma0114 apoprotein, the binary Sma0114·Ca(2+) complex, and the ternary Sma0114·Ca(2+)·BeF3(-) complex. The loss of entropy due to ligand binding at the active site is compensated by increased flexibility in the 455 face. The dynamic character of the 455 face in Sma0114, which results in part from the replacement of helix α4 by a flexible loop, may facilitate induced-fit recognition of target molecules.

Better to light a candle than curse the darkness: illuminating spatial localization and temporal dynamics of rapid microbial growth in the rhizosphere.

The rhizosphere is a hotbed of microbial activity in ecosystems, fueled by carbon compounds from plant roots. Basic questions about the location and dynamics of plant-spurred microbial growth in the rhizosphere are difficult to answer with standard, destructive soil assays mixing a multitude of microbe-scale microenvironments in a single, often sieved, sample. Soil microbial biosensors designed with the luxCDABE reporter genes fused to a promoter of interest enable continuous imaging of the microbial perception of (and response to) environmental conditions in soil. We used the common soil bacterium Pseudomonas putida KT2440 as host to plasmid pZKH2 containing a fusion between the strong constitutive promoter nptII and luxCDABE (coding for light-emitting proteins) from Vibrio fischeri. Experiments in liquid media demonstrated that high light production by KT2440/pZKH2 was associated with rapid microbial growth supported by high carbon availability. We applied the biosensors in microcosms filled with non-sterile soil in which corn (Zea mays L.), black poplar (Populus nigra L.), or tomato (Solanum lycopersicum L.) was growing. We detected minimal light production from microbiosensors in the bulk soil, but biosensors reported continuously from around roots for as long as six days. For corn, peaks of luminescence were detected 1-4 and 20-35 mm along the root axis behind growing root tips, with the location of maximum light production moving farther back from the tip as root growth rate increased. For poplar, luminescence around mature roots increased and decreased on a coordinated diel rhythm, but was not bright near root tips. For tomato, luminescence was dynamic, but did not exhibit a diel rhythm, appearing in acropetal waves along roots. KT2440/pZKH2 revealed that root tips are not always the only, or even the dominant, hotspots for rhizosphere microbial growth, and carbon availability is highly variable in space and time around roots.

Nuclear magnetic resonance structure and dynamics of the response regulator Sma0114 from Sinorhizobium meliloti.

Receiver domains control intracellular responses triggered by signal transduction in bacterial two-component systems. Here, we report the solution nuclear magnetic resonance structure and dynamics of Sma0114 from the bacterium Sinorhizobium meliloti, the first such characterization of a receiver domain from the HWE-kinase family of two-component systems. The structure of Sma0114 adopts a prototypical α(5)/ß(5) Rossman fold but has features that set it apart from other receiver domains. The fourth ß-strand of Sma0114 houses a PFxFATGY sequence motif, common to many HWE-kinase-associated receiver domains. This sequence motif in Sma0114 may substitute for the conserved Y-T coupling mechanism, which propagates conformational transitions in the 455 (α4-ß5-α5) faces of receiver domains, to prime them for binding downstream effectors once they become activated by phosphorylation. In addition, the fourth α-helix of the consensus 455 face in Sma0114 is replaced with a segment that shows high flexibility on the pico- to nanosecond time scale by (15)N relaxation data. Secondary structure prediction analysis suggests that the absence of helix α4 may be a conserved property of the HWE-kinase-associated family of receiver domains to which Sma0114 belongs. In spite of these differences, Sma0114 has a conserved active site, binds divalent metal ions such as Mg(2+) and Ca(2+) that are required for phosphorylation, and exhibits micro- to millisecond active-site dynamics similar to those of other receiver domains. Taken together, our results suggest that Sma0114 has a conserved active site but differs from typical receiver domains in the structure of the 455 face that is used to effect signal transduction following activation.

NMR assignments for the Sinorhizobium meliloti response regulator Sma0114.

Response regulators are terminal ends of bacterial two-component systems that undergo extensive structural reorganization in response to phosphoryl transfer from their cognate histidine kinases. The response regulator encoded by the gene sma0114 of Sinorhizobium meliloti is a part of a unique class of two-component systems that employ HWE histidine kinases. The distinct features of Sma0114 include a PFxFATGY motif that houses the conserved threonine in the "Y-T coupling" conformational switch which mediates output response through downstream protein-protein interactions, and the replacement of the conserved phenylalanine/tyrosine in Y-T coupling by a leucine. Here we present (1)H, (15)N, and (13)C NMR assignments for Sma0114. We identify the secondary structure of the protein based on TALOS chemical shift analysis, (3)J(HNHα) coupling constants and hydrogen-deuterium exchange. The secondary structure determined by NMR is in good agreement with that predicted from the sequence. Both methods suggest that Sma0114 differs from standard CheY-like folds by missing the fourth α-helix. Our initial NMR characterization of Sma0114 paves the way to a full investigation of the structure and dynamics of this response regulator.

Characterization of a two-component regulatory system that regulates succinate-mediated catabolite repression in Sinorhizobium meliloti.

When they are available, Sinorhizobium meliloti utilizes C(4)-dicarboxylic acids as preferred carbon sources for growth while suppressing the utilization of some secondary carbon sources such as α- and ß-galactosides. The phenomenon of using succinate as the sole carbon source in the presence of secondary carbon sources is termed succinate-mediated catabolite repression (SMCR). Genetic screening identified the gene sma0113 as needed for strong SMCR when S. meliloti was grown in succinate plus lactose, maltose, or raffinose. sma0113 and the gene immediately downstream, sma0114, encode the proteins Sma0113, an HWE histidine kinase with five PAS domains, and Sma0114, a CheY-like response regulator lacking a DNA-binding domain. sma0113 in-frame deletion mutants show a relief of catabolite repression compared to the wild type. sma0114 in-frame deletion mutants overproduce polyhydroxybutyrate (PHB), and this overproduction requires sma0113. Sma0113 may use its five PAS domains for redox level or energy state monitoring and use that information to regulate catabolite repression and related responses.

Micro-scale water potential gradients visualized in soil around plant root tips using microbiosensors.

Water availability and movement in soil are critical determinants of resource availability to, and interactions among, members of the soil community. However, it has been impossible to observe gradients in soil water potential empirically at millimetre spatial scales. Here we describe progress towards that goal using output from two microbial biosensors, Pantoea agglomerans BRT98/pPProGreen and Pseudomonas putida KT2442/pPProGreen, engineered with a reporter system based on the osmotically sensitive proU promoter from Escherichia coli. The proU-GFP construct in both microbiosensors produced green fluorescent protein (GFP) as a function total water potential in nonsterile soil. Controlled experiments in liquid culture showed that dramatically different microbiosensor growth rates (resulting from exposure to different salts as osmolytes) did not alter the GFP output as a function of water potential in either sensor, but P. agglomerans' GFP levels at a given water potential were strongly influenced by the type of carbon (energy) source available to the microbes. In non-sterile rhizosphere soil along Zea mays L. roots, though GFP expression was quite variable, microbiosensors reported statistically significantly more negative soil water potentials as a function of axial distance from root tips, reflecting the gradient in soil water potential hypothesized to develop during transpiration.

Plasmids that insert into the rhamnose utilization locus, rha: a versatile tool for genetic studies in Sinorhizobium meliloti.

Described is a suite of plasmids that can be used to deliver DNA into a specific site in the chromosome of Sinorhizobium meliloti with a minimal impact in the physiology of the organism. This allows stable, single-copy, insertions of DNA while maintaining a constant chromosomal context. The plasmids integrate into rhaS, one of a group of genes encoding proteins for rhamnose utilization, enabling a simple screening method for recombinants, while leaving other cellular processes unaffected by the insertion. Construction of plasmids for gfp labeling, mutant complementation, gene expression and protein over-expression studies are outlined. The rha insertion plasmids constitute a flexible and easy to use collection of cloning vectors that can be efficiently delivered by conjugation or transformation, and that could be used in genetic and physiology studies of S. meliloti, and, with minor modifications, in other bacterial species.