Inhibition of Microbial Growth and Biofilm Formation in Pure and Mixed Bacterial Samples.

Hydraulic fracturing, or fracking, requires large amounts of water to extract fossil fuel from rock formations. As a result of hydraulic fracturing, the briny wastewater, often termed back-produced fracturing or fracking water (FW), is pumped into holding ponds. One of the biggest challenges with produced water management is controlling microbial activity that could reduce the pond water's reusable layer and pose a significant environmental hazard. This study focuses on the characterization of back-produced water that has been hydraulically fractured using chemical and biological analysis and the development of a high-throughput screening method to evaluate and predict the antimicrobial effect of four naturally and commercially available acidic inhibitors (edetic acid, boric acid, tannic acid, and lactic acid) on the growth of the FW microbiome. Liquid cultures and biofilms of two laboratory model strains, the vegetative Escherichia coli MG1655, and the spore-forming Bacillus atrophaeus (also known as Bacillus globigii, BG) bacteria, were used as reference microorganisms. Planktonic bacteria in FW were more sensitive to antimicrobials than sessile bacteria in biofilms. Spore-forming BG bacteria exhibited more sensitivity to acidic inhibitors than the vegetative E. coli cells. Organic acids were the most effective bacterial growth inhibitors in liquid culture and biofilm.

Escherichia coli resistance mechanism AcrAB-TolC efflux pump interactions with commonly used antibiotics: a molecular dynamics study.

While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism of Escherichia coli (E. coli) cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand-protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand-protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showing E. coli cells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms.

Binding behavior of receptor binding domain of the SARS-CoV-2 virus and ivermectin.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), sparked an international debate on effective ways to prevent and treat the virus. Specifically, there were many varying opinions on the use of ivermectin (IVM) throughout the world, with minimal research to support either side. IVM is an FDA-approved antiparasitic drug that was discovered in the 1970s and was found to show antiviral activity. The objective of this study is to examine the binding behavior and rates of association and dissociation between SARS-CoV-2 receptor binding domain (RBD), IVM, and their combination using aminopropylsilane (APS) biosensors as surrogates for the hydrophobic interaction between the viral protein and human angiotensin-converting enzyme 2 (ACE2) receptors to determine the potential of IVM as a repurposed drug for SARS-CoV-2 prevention and treatment. The IVM, RBD, and combination binding kinetics were analyzed using biolayer interferometry (BLI) and validated with multiple in silico techniques including protein-ligand docking, molecular dynamics simulation, molecular mechanics-generalized Born surface area (MM-GBSA), and principal component analysis (PCA). Our results suggest that with increasing IVM concentrations the association rate with the hydrophobic biosensor increases with a simultaneous decrease in dissociation. Significant kinetic changes to RBD, when combined with IVM, were found only at a concentration a thousand times the approved dosage with minimal changes found over a 35-min time period. Our study suggests that IVM is not an effective preventative or treatment method at the currently approved dosage.

Oversampling methods for machine learning model data training to improve model capabilities to predict the presence of Escherichia coli MG1655 in spinach wash water.

We assessed the efficacy of oversampling techniques to enhance machine learning model performance in predicting Escherichia coli MG1655 presence in spinach wash water. Three oversampling methods were applied to balance two datasets, forming the basis for training random forest (RF), support vector machines (SVMs), and binomial logistic regression (BLR) models. Data underwent method-specific centering and standardization, with outliers replaced by feature-specific means in training datasets. Testing occurred without these preprocessing steps. Model hyperparameters were optimized using a subset of testing data via 10-fold cross-validation. Models were trained on full datasets and tested on newly acquired spinach wash water samples. Synthetic Minority Oversampling Technique (SMOTE) and Adaptive Synthetic Sampling approach (ADASYN) achieved strong results, with SMOTE RF reaching an accuracy of 90.0%, sensitivity of 93.8%, specificity of 87.5%, and an area under the curve (AUC) of 98.2% (without data preprocessing) and ADASYN achieving 86.55% accuracy, 87.5% sensitivity, 83.3% specificity, and a 92.4% AUC. SMOTE and ADASYN significantly improved (p < 0.05) SVM and RF models, compared to their non-oversampled counterparts without preprocessing. Data preprocessing had a mixed impact, improving (p < 0.05) the accuracy and specificity of the BLR model but decreasing the accuracy and specificity (p < 0.05) of the SVM and RF models. The most influential physiochemical feature for E. coli detection in wash water was water conductivity, ranging from 7.9 to 196.2 µS. Following closely was water turbidity, ranging from 2.97 to 72.35 NTU within this study.

The Effects of Airflow on the Mechanosensitive Channels of Escherichia coli MG1655 and the Impact of Survival Mechanisms Triggered.

Understanding how bacteria respond to ventilated environments is a crucial concept, especially when considering accurate airflow modeling and detection limits. To properly design facilities for aseptic conditions, we must minimize the parameters for pathogenic bacteria to thrive. Identifying how pathogenic bacteria continue to survive, particularly due to their multi-drug resistance characteristics, is necessary for designing sterile environments and minimizing pathogen exposure. A conserved characteristic among bacterial organisms is their ability to maintain intracellular homeostasis for survival and growth in hostile environments. Mechanosensitive (MS) channels are one of the characteristics that guide this phenomenon. Interestingly, during extreme stress, bacteria will forgo favorable homeostasis to execute fast-acting survival strategies. Physiological sensors, such as MS channels, that trigger this survival mechanism are not clearly understood, leaving a gap in how bacteria translate physical stress to an intracellular response. In this paper, we study the role of mechanosensitive ion channels that are potentially triggered by aerosolization. We hypothesize that change in antimicrobial uptake is affected by aerosolization stress. Bacteria regulate their defense mechanisms against antimicrobials, which leads to varying susceptibility. Based on this information we hypothesize that aerosolization stress affects the antimicrobial resistance defense mechanisms of Escherichia coli (E. coli). We analyzed the culturability of knockout E. coli strains with different numbers of mechanosensitive channels and compared antibiotic susceptibility under stressed and unstressed airflow conditions. As a result of this study, we can identify how the defensive mechanisms of resistant bacteria are triggered for their survival in built environments. By changing ventilation airflow velocity and observing the change in antibiotic responses, we show how pathogenic bacteria respond to ventilated environments via mechanosensitive ion channels.

Sampling and Characterization of Bioaerosols in Poultry Houses.

Two poultry Confined Animal Feeding Units (CAFUs), "House A" and "House B", were selected from the TAMU poultry facility for the study, and samples were collected over a five-day period. Bioaerosol sampling was conducted using a Wetted Wall Cyclone (WWC) bioaerosol collector at the two CAFU houses, in which House A housed approximately 720 broiler chickens and roosters, while House B remained unoccupied and served as a reference. Both houses consisted of 24 pens arranged on either side of a central walkway. Bacterial content analysis was conducted using microbial plating, real-time Polymerase Chain Reaction (PCR), and Fatty Acid Methyl Ester (FAME) analysis, while ambient temperature and relative humidity were also monitored. The concentrations of microorganisms in House A showed a highly dynamic range, ranging from 4000 to 60,000 colony forming units (CFU) per cubic meter of air. Second, the WWC samples contained approximately ten-fold more bacterial DNA than the filter samples, suggesting higher levels of viable cells captured by the WWC. Third, significant concentrations of pathogens, including Salmonella, Staphylococcus, and Campylobacter, were detected in the poultry facility. Lastly, the WWC system demonstrated effective functionality and continuous operation, even in the challenging sampling environment of the CAFU. The goal of this study was to characterize the resident population of microorganisms (pathogenic and non-pathogenic) present in the CAFUs and to evaluate the WWC's performance in such an environment characterized by elevated temperature, high dust content, and feathers. This knowledge could then be used to improve understanding microorganism dynamics in CAFUs including the spread of bacterial infections between animals and from animals to humans that work in these facilities, as well as of the WWC performance in this type of environment (elevated temperature, high content of dust and feathers). A more comprehensive understanding can aid in improving the management of bacterial infections in these settings.

Dispersion of sneeze droplets in a meat facility indoor environment - Without partitions.

Spreading patterns of the coronavirus disease (COVID-19) showed that infected and asymptotic carriers both played critical role in escalating transmission of virus leading to global pandemic. Indoor environments of restaurants, classrooms, hospitals, offices, large assemblies, and industrial installations are susceptible to virus outbreak. Industrial facilities such as fabrication rooms of meat processing plants, which are laden with moisture and fat in indoor air are the most sensitive spaces. Fabrication room workers standing next to each other are exposed to the risk of long-range viral droplets transmission within the facility. An asymptomatic carrier may transmit the virus unintentionally to fellow workers through sporadic sneezing leading to community spread. A novel Computational Fluid Dynamics (CFD) model of a fabrication room with typical interior (stationary objects) was prepared and investigated. Study was conducted to identify indoor airflow patterns, droplets spreading patterns, leading droplets removal mechanism, locations causing maximum spread of droplets, and infection index for workers along with stationary objects in reference to seven sneeze locations covering the entire room. The role of condensers, exhaust fans and leakage of indoor air through large and small openings to other rooms was investigated. This comprehensive study presents flow scenarios in the facility and helps identify locations that are potentially at lower or higher risk for exposure to COVID-19. The results presented in this study are suitable for future engineering analyses aimed at redesigning public spaces and common areas to minimize the spread of aerosols and droplets that may contain pathogens.

Quiescence of Escherichia coli Aerosols to Survive Mechanical Stress during High-Velocity Collection.

A low cutpoint wetted wall bioaerosol sampling cyclone (LCP-WWC), with an aerosol sampling flow rate of 300 L/min at 55″ H2O pressure drop and a continuous liquid outflow rate of about 0.2 mL/min, was developed by upgrading an existing system. The laboratory strain Escherichia coli MG1655 was aerosolized using a six-jet Collison Nebulizer and collected at high velocity using the LCP-WWC for 10 min with different collection liquids. Each sample was quantitated during a 15-day archiving period after aerosolization for culturable counts (CFUs) and gene copy numbers (GCNs) using microbial plating and whole-cell quantitative polymerase chain (qPCR) reaction. The samples were analyzed for protein composition and antimicrobial resistance using protein gel electrophoresis and disc diffusion susceptibility testing. Aerosolization and collection were followed by an initial period of quiescence or dormancy. After 2 days of archiving at 4 °C and RT, the bacteria exhibited increased culturability and antibiotic resistance (ABR), especially to cell wall inhibitors (ampicillin and cephalothin). The number of resistant bacteria on Day 2 increased nearly four-times compared to the number of cells at the initial time of collection. The mechanical stress of aerosolization and high-velocity sampling likely stunned the cells triggering a response of dormancy, though with continued synthesis of vital proteins for survival. This study shows that an increase in intensity in environmental conditions surrounding airborne bacteria affects their ability to grow and their potential to develop antimicrobial resistance.

Temporal Variation of SARS-CoV-2 Levels in Wastewater from a Meat Processing Plant.

Wastewater-based surveillance (WBS) on SARS-CoV-2 has been proved to be an effective approach to estimate the prevalence of COVID-19 in communities and cities. However, its application was overlooked at smaller scale, such as a single facility. Meat processing plants are hotspots for COVID-19 outbreaks due to their unique environment that are favorable for the survival and persistence of SARS-CoV-2. This is the first known WBS study in meat processing plants. The goal was to understand the temporal variation of the SARS-CoV-2 levels in wastewater from a meat processing plant in Canada during a three-month campaign and to find any correlation with clinically confirmed cases in the surrounding city area. Higher SARS-CoV-2 concentrations and detection frequencies were observed in the solid fraction compared to the liquid fraction of the wastewater. The viruses can be preserved in the solid fraction of wastewater for up to 12 days. The wastewater virus level did not correlate to the city-wide COVID-19 cases due to the unmatching scales. WBS on SARS-CoV-2 in meat processing plants can be useful for identifying COVID-19 outbreaks in the facility and serve as an effective alternative when resources for routine individual testing are not available.

Numerical investigation on indoor environment decontamination after sneezing.

More than 320 million people worldwide were affected by SARS-CoV-2 or COVID-19, which already caused more than 5.5 million deaths. COVID-19 spreads through air when an infected person breathes, coughs, or sneezes out droplets containing virus. Emerging variants like Omicron with positivity rate of 16 (highest among others) present a greater risk of virus spread, so all types of indoor environments become critically important. Strategically adopted Heating Ventilation and Air Conditioning (HVAC) approach can significantly reduce the virus spread by early removal of contaminated aerosolized droplets. We modeled different HVAC configurations to characterize the diffusion of contaminated droplets cloud through Computational Fluid Dynamics (CFD) simulations of sneeze in standard hospital room as indoor scenario. Injection of saliva droplets with characteristics of exhaled air from lungs was applied to mimic real sneeze. CFD simulations have been performed for three HVAC configurations at two Air Change per Hour (ACH) rates; 6 and 15 ACH. For the first time, use of air curtain at low flow rate has been examined. Simulations provide high fidelity spatial and temporal droplets cloud diffusion under different HVAC configurations, showing spread in room indoor environment up to 360 s. Over 92% of ejected sneeze mass is removed from room air within seconds while the remaining 8% or less becomes airborne with droplets (<50 µm size) and tends to spread uniformly with regular HVAC configuration. Low-speed air curtain accelerates decontamination by efficiently removing aerosolized 1-50 µm size droplets. Study investigates role of droplets removal mechanisms such as escape, evaporation, and deposition on surfaces. Interestingly, results show presence of contaminated droplets even after 5 min of sneeze, which can be effectively removed using low-speed air curtain. Study finds that high ventilation rate requirements can be optimized to modify earlier and new hospital designs to reduce the spread of airborne disease.

Environmental Effects on Viable Virus Transport and Resuspension in Ventilation Airflow.

To understand how SARS-CoV-2 spreads indoors, in this study bovine coronavirus was aerosolized as simulant into a plexiglass chamber with coupons of metal, wood and plastic surfaces. After aerosolization, chamber and coupon surfaces were swiped to quantify the virus concentrations using quantitative polymerase chain reaction (qPCR). Bio-layer interferometry showed stronger virus association on plastic and metal surfaces, however, higher dissociation from wood in 80% relative humidity. Virus aerosols were collected with the 100 L/min wetted wall cyclone and the 50 L/min MD8 air sampler and quantitated by qPCR. To monitor the effect of the ventilation on the virus movement, PRD1 bacteriophages as virus simulants were disseminated in a ¾ scale air-conditioned hospital test room with twelve PM2.5 samplers at 15 L/min. Higher virus concentrations were detected above the patient's head and near the foot of the bed with the air inlet on the ceiling above, exhaust bottom left on the wall. Based on room layout, air measurements and bioaerosol collections computational flow models were created to visualize the movement of the virus in the room airflow. The addition of air curtain at the door minimized virus concentration while having the inlet and exhaust on the ceiling decreased overall aerosol concentration. Controlled laboratory experiments were conducted in a plexiglass chamber to gain more insight into the fundamental behavior of aerosolized SARS-CoV-2 and understand its fate and transport in the ambient environment of the hospital room.

Binding behavior of spike protein and receptor binding domain of the SARS-CoV-2 virus at different environmental conditions.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of the COVID-19 pandemic that originated in China in December 2019. Although extensive research has been performed on SARS-CoV-2, the binding behavior of spike (S) protein and receptor binding domain (RBD) of SARS-CoV-2 at different environmental conditions have yet to be studied. The objective of this study is to investigate the effect of temperature, fatty acids, ions, and protein concentration on the binding behavior and rates of association and dissociation between the S protein and RBD of SARS-CoV-2 and the hydrophobic aminopropylsilane (APS) biosensors using biolayer interferometry (BLI) validated with molecular dynamics simulation. Our results suggest three conditions-high ionic concentration, presence of hydrophobic fatty acids, and low temperature-favor the attachment of S protein and RBD to hydrophobic surfaces. Increasing the temperature within an hour from 0 to 25 °C results in S protein detachment, suggesting that freezing can cause structural changes in the S protein, affecting its binding kinetics at higher temperature. At all the conditions, RBD exhibits lower dissociation capabilities than the full-length S trimer protein, indicating that the separated RBD formed stronger attachment to hydrophobic surfaces compared to when it was included in the S protein.

Ternary Fingerprints with Reference Odor for Fluctuation-Enhanced Sensing.

An improved method for fluctuation-enhanced sensing (FES) is introduced. We enhanced the old binary fingerprinting method, where the fingerprint bit values were ±1, by introducing ternary fingerprint bits utilizing a reference odor. In the ternary method, the fingerprint bit values are -1, 0, and +1, where the 0 value stands for the situation where the slope of the spectrum is identical to that of the reference odor. The application of the reference odor spectrum makes the fingerprint relative to the reference. The ternary nature and the reference feature increase the information entropy of the fingerprints. The method is briefly illustrated by sensing bacterial odor in cow manure isolates.

Design of thermal wind sensor with constant power control and wind vector measurement method.

This paper presents a conceptual wind vector detector for measuring the velocity and direction of wind in enclosed or semi-enclosed large spaces. Firstly, a thermal wind sensor with constant power control was manufactured and then used as a wind velocity sensing unit. Secondly, a sensor bracket equipped with three thermal wind sensors was designed, the fluid dynamic response regularity of the measured wind field to the sensor bracket was analyzed using ANSYS Fluent CFD software, and then its structural parameters were optimized to improve measurement accuracy. The sensor bracket was fabricated via 3D printing. Finally, a unique wind vector measurement method was developed for the wind vector detector. Experimental results showed that the measured velocity range of the thermal wind sensor satisfied the requirements of being within 0-15 m/s with an accuracy of ±0.3 m/s, and the wind direction angle range of the wind vector detector was within 0-360° with an accuracy of ±5°. By changing the applied power control value of the thermal wind sensor and structural parameters of the sensor bracket, the measurement range and accuracy of the wind vector detector can be adjusted to suit different applications.

Detection of influenza A virus from agricultural fair environment: Air and surfaces.

Agricultural fairs facilitate an environment conducive to the spread of influenza A virus with large numbers of pigs from various different locales comingling for several days (5-8 days). Fairs are also associated with zoonotic transmission of influenza A virus as humans have unrestricted contact with potentially infected swine throughout the fair's duration. Since 2005, the Centers for Disease Control and Prevention has reported 468 cases of variant influenza A virus, with most cases having had exposure to swine at agricultural fairs. Many mechanisms have been proposed as potential direct and indirect routes of transmission that may be enhancing intra- and inter-species transmission of influenza A virus at fairs. This study examines airborne respiratory droplets and portable animal-care items as potential routes of transmission that may be contributing to enhanced viral spread throughout the swine barn and the resulting variant cases of influenza A. Air samples were taken from inside swine barns at 25 fairs between the years 2013 and 2014. Influenza A virus was detected molecularly in 11 of 59 (18.6%) air samples, representing 4 of the 25 fairs. Viable H1N1 virus, matching virus recovered from swine at the fair, was recovered from the air at one fair in 2013. During the summer of 2016, 75 of 400 (18.8%) surface samples tested positive for molecular presence of influenza A virus and represented 10 of 20 fairs. Seven viral isolates collected from four fairs were recovered from the surfaces. Whole genome sequences of the viruses recovered from the surfaces are >99% identical to the viruses recovered from individual pigs at each respective fair. The detection and recovery of influenza A virus from both the air and surfaces found within the swine barn at agricultural fairs provide evidence for potential viral transmission through these routes, which may contribute to both intra- and inter-species transmission, threatening public health. These findings reinforce the need for new and improved mitigation strategies at agricultural fairs in order to reduce the risk to animal and public health.

Binary fingerprints at fluctuation-enhanced sensing.

We have developed a simple way to generate binary patterns based on spectral slopes in different frequency ranges at fluctuation-enhanced sensing. Such patterns can be considered as binary "fingerprints" of odors. The method has experimentally been demonstrated with a commercial semiconducting metal oxide (Taguchi) sensor exposed to bacterial odors (Escherichia coli and Anthrax-surrogate Bacillus subtilis) and processing their stochastic signals. With a single Taguchi sensor, the situations of empty chamber, tryptic soy agar (TSA) medium, or TSA with bacteria could be distinguished with 100% reproducibility. The bacterium numbers were in the range of 2.5 × 10(4)-10(6). To illustrate the relevance for ultra-low power consumption, we show that this new type of signal processing and pattern recognition task can be implemented by a simple analog circuitry and a few logic gates with total power consumption in the microWatts range.

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex.

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.

Microbial products trigger amino acid exudation from plant roots.

Plants naturally cycle amino acids across root cell plasma membranes, and any net efflux is termed exudation. The dominant ecological view is that microorganisms and roots passively compete for amino acids in the soil solution, yet the innate capacity of roots to recover amino acids present in ecologically relevant concentrations is unknown. We find that, in the absence of culturable microorganisms, the influx rates of 16 amino acids (each supplied at 2.5 microm) exceed efflux rates by 5% to 545% in roots of alfalfa (Medicago sativa), Medicago truncatula, maize (Zea mays), and wheat (Triticum aestivum). Several microbial products, which are produced by common soil microorganisms such as Pseudomonas bacteria and Fusarium fungi, significantly enhanced the net efflux (i.e. exudation) of amino acids from roots of these four plant species. In alfalfa, treating roots with 200 microm phenazine, 2,4-diacetylphloroglucinol, or zearalenone increased total net efflux of 16 amino acids 200% to 2,600% in 3 h. Data from (15)N tests suggest that 2,4-diacetylphloroglucinol blocks amino acid uptake, whereas zearalenone enhances efflux. Thus, amino acid exudation under normal conditions is a phenomenon that probably reflects both active manipulation and passive uptake by microorganisms, as well as diffusion and adsorption to soil, all of which help overcome the innate capacity of plant roots to reabsorb amino acids. The importance of identifying potential enhancers of root exudation lies in understanding that such compounds may represent regulatory linkages between the larger soil food web and the internal carbon metabolism of the plant.

Roles for riboflavin in the Sinorhizobium-alfalfa association.

Genes contributing to riboflavin production in Sinorhizobium meliloti were identified, and bacterial strains that overproduce this vitamin were constructed to characterize how additional riboflavin affects interactions between alfalfa (Medicago sativa) and S. meliloti. Riboflavin-synthesis genes in S. meliloti were found in three separate linkage groups and designated as ribBA, ribDribC, and ribH for their similarities to Escherichia coli genes. The ribBA and ribC loci complemented corresponding E. coli rib mutants. S. meliloti cells containing extra copies of ribBA released 10 to 20% more riboflavin than a control strain but grew at similar rates in a defined medium lacking riboflavin. Cells carrying extra copies of ribBA colonized roots to densities that were 55% higher than that of a control strain. No effect of extra rib genes was detected on alfalfa grown in the absence or presence of combined N. These results support the importance of extracellular riboflavin for alfalfa root colonization by S. meliloti and are consistent with the hypothesis that this molecule benefits bacteria indirectly through an effect on the plant.