Bioengineered chitosan-magnesium nanocomposite: A novel agricultural antimicrobial agent against Acidovorax oryzae and Rhizoctonia solani for sustainable rice production.

Chitosan is a potent biopolymer having promising antimicrobial properties against phytopathogens. Recently, engineered nanomaterials (ENMs) have gained much attention due to their potential application in the plant disease management. In this study, we reported the green synthesis of chitosan-magnesium (CS-Mg) nanocomposite and its antimicrobial activity against two rice pathogens namely Acidovorax oryzae and Rhizoctonia solani for the first time. The green MgO nanoparticles synthesized by using a native Bacillus sp. strain RNT3, were used to fabricate CS-Mg nanocomposite utilizing one-pot synthesis method. The synthesis of CS-Mg nanocomposite was further confirmed by using UV-vis spectroscopy, whereas, FTIR and XRD analysis showed the capping of CS-Mg nanocomposites by different functional groups together with their crystalline structure, respectively. Besides, SEM and TEM images revealed the spherical shape along with the particles size ranging from 29 to 60 nm. Moreover, EDS analysis confirmed the elemental purity of nanocomposite. The CS-Mg nanocomposite showed remarkable antimicrobial activity against A. oryzae and R. solani and significantly inhibited the growth as compared to non-treated control. The ultrastructure studies showed damaged structure of cell wall and internal cellular organelles after treatment with 100 µg mL-1 CS-Mg nanocomposite. The results of this study indicated that CS-Mg nanocomposite-based antimicrobial agents could be considered as promising nanopesticides against phytopathogens in plant disease management.

Antimicrobial activities of organic acid vapors against Acidovorax citrulli, Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes on Cucurbitaceae seeds.

This study investigated the antimicrobial activities of organic acid vapors against a phytopathogen (Acidovorax citrulli) and foodborne pathogens (Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes) on the surface of Cucurbitaceae seeds. Germination percentages of cucumber, honeydew melon and watermelon seeds treated with acetic and propionic acid vapors (100 mg/L) at 50 °C and 43% or 85% relative humidity (RH) for up to 2 h did not significantly (P > 0.05) decrease. Treatment with formic acid significantly (P ≤ 0.05) decreased the germination percentage. The antimicrobial activities of acetic and propionic acid vapors (100 mg/L; 50 °C; 43% or 85% RH) were determined. A. citrulli was inactivated within 1 h on cucumber and watermelon seeds, regardless of type of organic acid or RH. The phytopathogen was reduced to levels below the detection limit (-0.5 log CFU/g) for enrichment on honeydew melon seeds treated with acetic acid vapor. S. enterica and L. monocytogenes were inactivated within 2 h at 85% RH on honeydew melon and watermelon seeds treated with acetic acid and propionic acid vapors. E. coli O157: H7 was inactivated by treatment with acetic acid vapor at 85% RH. This study provides useful information for developing a method to decontaminate Curcurbitaceae seeds using organic acid vapors as lethal agents.

Analysis of the influence of flame sterilization included in sampling operations on shake-flask cultures of microorganisms.

Shake-flask cultures of microorganisms involve flame sterilization during sampling, which produces combustion gas with high CO2 concentrations. The gaseous destination has not been deeply analyzed. Our aim was to investigate the effect of flame sterilization on the headspace of the flask and on the shake-flask culture. In this study, the headspace CO2 concentration was found to increase during flame sterilization ~0.5-2.0% over 5-20 s empirically using the Circulation Direct Monitoring and Sampling System. This CO2 accumulation was confirmed theoretically using Computational Fluid Dynamics; it was 9% topically. To evaluate the influence of CO2 accumulation without interference from other sampling factors, the flask gas phase formed by flame sterilization was reproduced by aseptically supplying 99.8% CO2 into the headspace, without sampling. We developed a unit that can be sampled in situ without interruption of shaking, movement to a clean bench, opening of the culture-plug, and flame sterilization. We observed that the growth behaviour of Escherichia coli, Pelomonas saccharophila, Acetobacter pasteurianus, and Saccharomyces cerevisiae was different depending on the CO2 aeration conditions. These results are expected to contribute to improving microbial cell culture systems.

Controlled Crystallization and Transformation of Carbonate Minerals with Dumbbell-like Morphologies on Bacterial Cell Templates.

Research on the biogenic-specific polymorphism and morphology of carbonate has been gaining momentum in the fields of biomineralization and industrial engineering in recent years. We report the nucleation of carbonate particles on bacterial cell templates to produce a novel dumbbell-like morphology which was assembled by needle-like crystals of magnesium calcite or aragonite radiating out from both ends of the template bacterium. Mature dumbbell-like structures had a tendency to break apart in the central template region, which was made up mostly of weak amorphous carbonate. Further crystal growth, especially at the template region, transformed the broken pieces into spherulites. Rod-like cell templates were essential for the formation of dumbbell-like morphologies, and we propose a possible formation mechanism of the dumbbell-like morphology. Our findings provide new perspectives on the morphological formation mechanism in biomineralization systems and may have a potential significance in assembling composite materials suitable for industrial applications.

Casamino acids slow motility and stimulate surface growth in an extreme oligotroph.

Environmental cues that regulate motility are poorly understood, but specific carbon and nitrogen sources, such as casamino acids (CAA), are known to stimulate motility in model organisms. However, natural environments are commonly more nutrient-limited than laboratory growth media, and the effect of energy-rich CAA on the motility of oligotrophic microorganisms is unknown. In this study, an extreme oligocarbotroph, Variovorax paradoxus YC1, was isolated from weathered shale rock within a disused mine level in North Yorkshire, UK. The addition of 0.1% CAA to minimal media significantly reduced the motility of YC1 after 72 h and inhibited swimming motility resulting in enhanced surface growth. We propose this response to CAA is a physiological adaptation to oligotrophy, facilitating the colonization of nutrient-rich environments.

Exploiting Hydrogenophaga pseudoflava for aerobic syngas-based production of chemicals.

Gasification is a suitable technology to generate energy-rich synthesis gas (syngas) from biomass or waste streams, which can be utilized in bacterial fermentation processes for the production of chemicals and fuels. Established microbial processes currently rely on acetogenic bacteria which perform an energetically inefficient anaerobic CO oxidation and acetogenesis potentially hampering the biosynthesis of complex and ATP-intensive products. Since aerobic oxidation of CO is energetically more favorable, we exploit in this study the Gram-negative ß-proteobacterium Hydrogenophaga pseudoflava DSM1084 as novel host for the production of chemicals from syngas. We sequenced and annotated the genome of H. pseudoflava and established a genetic engineering toolbox, which allows markerless chromosomal modification via the pk19mobsacB system and heterologous gene expression on pBBRMCS2-based plasmids. The toolbox was extended by identifying strong endogenous promotors such as PgapA2 which proved to yield high expression under heterotrophic and autotrophic conditions. H. pseudoflava showed relatively fast heterotrophic growth in complex and minimal medium with sugars and organic acids which allows convenient handling in lab routines. In autotrophic bioreactor cultivations with syngas, H. pseudoflava exhibited a growth rate of 0.06 h-1 and biomass specific uptakes rates of 14.2 ±â€¯0.3 mmol H2 gCDW-1 h-1, 73.9 ±â€¯1.8 mmol CO gCDW-1 h-1, and 31.4 ±â€¯0.3 mmol O2 gCDW-1 h-1. As proof of concept, we engineered the carboxydotrophic bacterium for the aerobic production of the C15 sesquiterpene (E)-α-bisabolene from the C1 carbon source syngas by heterologous expression of the (E)-α-bisabolene synthase gene agBIS. The resulting strain H. pseudoflava (pOCEx1:agBIS) produced 59 ±â€¯8 µg (E)-α-bisabolene L-1 with a volumetric productivity Qp of 1.2 ±â€¯0.2 µg L-1 h-1 and a biomass-specific productivity qp of 13.1 ±â€¯0.6 µg gCDW-1 h-1. The intrinsic properties and the genetic repertoire of H. pseudoflava make this carboxydotrophic bacterium a promising candidate for future aerobic production processes to synthesize more complex or ATP-intensive chemicals from syngas.

Development of a decontamination method to inactivate Acidovorax citrulli on Cucurbitaceae seeds without loss of seed viability.

BACKGROUND: Acidovorax citrulli is a plant pathogen causing bacterial fruit blotch in Cucurbitaceae family. Applying high concentration of disinfectants to seeds containing plant pathogen may substantially decrease the germination rate of seeds. Therefore, it is necessary to develop a hurdle technology which can inactivate plant pathogens without decreasing seed viability. This study was conducted to develop a decontamination method to inactivate the plant pathogen Acidovorax citrulli on Cucurbitaceae seeds by sequential treatments with aqueous chlorine dioxide (ClO2 ), drying, and dry heat. RESULTS: The maximum ClO2 concentration that did not lower germination rates of cucumber, honeydew melon, and watermelon seeds was ca. 100 µg mL-1 of ClO2 for 5 min. Optimal incubation conditions for drying seeds that had been treated with aqueous ClO2 were determined as 25 °C and 43% relative humidity (RH) for 48 h. The maximum dry-heat temperature that did not reduce germination rates of seeds, which had been treated with ClO2 and dried at 25 °C, was 60 °C at 43% RH for 24 h. When seeds containing A. citrulli (6.4-7.0 log CFU g-1 ) were treated with aqueous ClO2 (50 µg mL-1 , 5 min), dried (25 °C, 43% RH, 24 h), and dry heated (60 °C, 43% RH, 24 h), the pathogen was inactivated to below the detection limit from all three seed types (<-0.5 log CFU g-1 ). CONCLUSION: The decontamination conditions to inactivate A. citrullii from Cucurbitaceae seeds without decreasing the seed viability were determined (sequential treatment with ClO2 [50 µg mL-1 , 5 min], dried [25 °C, 43% RH, 24 h], and dry heated [60 °C, 43% RH, 24 h]). The results of this study may also be applicable to other plant pathogens on other types of seeds. © 2019 Society of Chemical Industry.

Role of type IV secretion system genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae strain RS-2.

Type IV secretion system (T4SS) is a specialized nanomachine that is utilized for the pathogenicity of gram-negative bacteria. However, the role of T4SS genes in virulence of rice bacterial brown stripe pathogen Acidovorax oryzae (Ao) strain RS-2 is not clear, which contains T4SS gene cluster based on genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type strain RS-2 and nine T4SS mutants, which were constructed in this study. Results indicated that mutation of pilT, pilM, pilQ, or pilZ3 genes not only significantly reduced bacterial virulence, but also caused a reduction of 20.4-62.0% in biofilm formation and 37.7-47.7% reduction in motility, but had no effect on exopolysaccharide (EPS) production or extracellular enzymatic activities when compared to the wild type. The four T4SS genes had a differential effect on bacterial growth after 24 h post-incubation. The complemented strains of the four T4SS mutants restored similar virulence symptom as the wild type. In addition, no change was observed in bacterial virulence by mutation of the other five T4SS genes. Totally, these results demonstrated that T4SS played vital roles in bacterial virulence, motility and biofilm formation in plant pathogen Ao strain RS-2.

L-Lysine-α-Oxidase: Acidovorax citrulli Bacterium Inhibitor.

Studies of the effects of Trichoderma harzianum Rifai F-180 culture fluid concentrate containing L-lysine-α-oxidase antitumor enzyme produced by the fungus and the homogenous enzyme, on ultrahazardous bacterium Acidovorax citrulli demonstrated the antibacterial activity of the concentrate. Trichoderma harzianum Rifai F-180 producing L-lysine-α-oxidase was cultured in a technological device at G. K. Skryabin Institute of Biochemistry and. Physiology of Microorganisms, Russian Academy of Sciences. Activity of L-lysine-α-oxidase in the resulted culture fluid concentrate was 0.54 U/ml, activity of the homogenous enzyme was 50 U/mg.

Multidisciplinary characterization of U(VI) sequestration by Acidovorax facilis for bioremediation purposes.

The contamination of the environment by U may affect plant life and consequently may have an impact on animal and human health. The present work describes U(VI) sequestration by Acidovorax facilis using a multidisciplinary approach combining wet chemistry, transmission electron microscopy, and spectroscopy methods (e.g. cryo-time resolved laser-induced fluorescence spectroscopy, extended X-ray absorption fine structure spectroscopy, and in-situ attenuated total reflection Fourier transform infrared spectroscopy). This bacterial strain is widely distributed in nature including U-contaminated sites. In kinetic batch experiments cells of A. facilis were contacted for 5 min to 48 h with 0.1 mM U(VI). The results show that the local coordination of U species associated with the cells depends upon time contact. U is bound mainly to phosphate groups of lipopolysaccharide (LPS) at the outer membrane within the first hour. And, that both, phosphoryl and carboxyl functionality groups of LPS and peptidoglycan of A. facilis cells may effectuate the removal of high U amounts from solution at 24-48 h of incubation. It is clearly demonstrated that A. facilis may play an important role in predicting the transport behaviour of U in the environment and that the results will contribute to the improvement of bioremediation methods of U-contaminated sites.

Role of the Genes of Type VI Secretion System in Virulence of Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae Strain RS-2.

The Type VI secretion system (T6SS) is a class of macromolecular machine that is required for the virulence of gram-negative bacteria. However, it is still not clear what the role of T6SS in the virulence of rice bacterial brown stripe pathogen Acidovorax avenae subsp. avenae (Aaa) is. The aim of the current study was to investigate the contribution of T6SS in Aaa strain RS2 virulence using insertional deletion mutation and complementation approaches. This strain produced weak virulence but contains a complete T6SS gene cluster based on a genome-wide analysis. Here we compared the virulence-related phenotypes between the wild-type (RS-2) and 25 T6SS mutants, which were constructed using homologous recombination methods. The mutation of 15 T6SS genes significantly reduced bacterial virulence and the secretion of Hcp protein. Additionally, the complemented 7 mutations ΔpppA, ΔclpB, Δhcp, ΔdotU, ΔicmF, ΔimpJ, and ΔimpM caused similar virulence characteristics as RS-2. Moreover, the mutant ΔpppA, ΔclpB, ΔicmF, ΔimpJ and ΔimpM genes caused by a 38.3~56.4% reduction in biofilm formation while the mutants ΔpppA, ΔclpB, ΔicmF and Δhcp resulted in a 37.5~44.6% reduction in motility. All together, these results demonstrate that T6SS play vital roles in the virulence of strain RS-2, which may be partially attributed to the reductions in Hcp secretion, biofilm formation and motility. However, differences in virulence between strain RS-1 and RS-2 suggest that other factors may also be involved in the virulence of Aaa.

Strain-specific consumption and transformation of alga-derived dissolved organic matter by members of the Limnohabitans-C and Polynucleobacter-B clusters of Betaproteobacteria.

We investigated changes in quality and quantity of extracellular and biomass-derived organic matter (OM) from three axenic algae (genera Rhodomonas, Chlamydomonas, Coelastrum) during growth of Limnohabitans parvus, Limnohabitans planktonicus and Polynucleobacter acidiphobus representing important clusters of freshwater planktonic Betaproteobacteria. Total extracellular and biomass-derived OM concentrations from each alga were approximately 20 mg l-1 and 1 mg l-1 respectively, from which up to 9% could be identified as free carbohydrates, polyamines, or free and combined amino acids. Carbohydrates represented 54%-61% of identified compounds of the extracellular OM from each alga. In biomass-derived OM of Rhodomonas and Chlamydomonas 71%-77% were amino acids and polyamines, while in that of Coelastrum 85% were carbohydrates. All bacteria grew on alga-derived OM of Coelastrum, whereas only Limnohabitans strains grew on OM from Rhodomonas and Chlamydomonas. Bacteria consumed 24%-76% and 38%-82% of all identified extracellular and biomass-derived OM compounds respectively, and their consumption was proportional to the concentration of each OM compound in the different treatments. The bacterial biomass yield was higher than the total identifiable OM consumption indicating that bacteria also utilized other unidentified alga-derived OM compounds. Bacteria, however, also produced specific OM compounds suggesting enzymatic polymer degradation or de novo exudation.

Sugarcane transcriptome analysis in response to infection caused by Acidovorax avenae subsp. avenae.

Sugarcane is an important tropical crop mainly cultivated to produce ethanol and sugar. Crop productivity is negatively affected by Acidovorax avenae subsp avenae (Aaa), which causes the red stripe disease. Little is known about the molecular mechanisms triggered in response to the infection. We have investigated the molecular mechanism activated in sugarcane using a RNA-seq approach. We have produced a de novo transcriptome assembly (TR7) from sugarcane RNA-seq libraries submitted to drought and infection with Aaa. Together, these libraries present 247 million of raw reads and resulted in 168,767 reference transcripts. Mapping in TR7 of reads obtained from infected libraries, revealed 798 differentially expressed transcripts, of which 723 were annotated, corresponding to 467 genes. GO and KEGG enrichment analysis showed that several metabolic pathways, such as code for proteins response to stress, metabolism of carbohydrates, processes of transcription and translation of proteins, amino acid metabolism and biosynthesis of secondary metabolites were significantly regulated in sugarcane. Differential analysis revealed that genes in the biosynthetic pathways of ET and JA PRRs, oxidative burst genes, NBS-LRR genes, cell wall fortification genes, SAR induced genes and pathogenesis-related genes (PR) were upregulated. In addition, 20 genes were validated by RT-qPCR. Together, these data contribute to a better understanding of the molecular mechanisms triggered by the Aaa in sugarcane and opens the opportunity for the development of molecular markers associated with disease tolerance in breeding programs.

Inhibitory Effect of Camptothecin against Rice Bacterial Brown Stripe Pathogen Acidovorax avenae subsp. avenae RS-2.

Camptothecin (CPT) has anticancer, antiviral, and antifungal properties. However, there is a dearth of information about antibacterial activity of CPT. Therefore, in this study, we investigated the inhibitory effect of CPT on Acidovorax avenae subsp. avenae strain RS-2, the pathogen of rice bacterial brown stripe, by measuring cell growth, DNA damage, cell membrane integrity, the expression of secretion systems, and topoisomerase-related genes, as well as the secretion of effector protein Hcp. Results indicated that CPT solutions at 0.05, 0.25, and 0.50 mg/mL inhibited the growth of strain RS-2 in vitro, while the inhibitory efficiency increased with an increase in CPT concentration, pH, and incubation time. Furthermore, CPT treatment affected bacterial growth and replication by causing membrane damage, which was evidenced by transmission electron microscopic observation and live/dead cell staining. In addition, quantitative real-time PCR analysis indicated that CPT treatment caused differential expression of eight secretion system-related genes and one topoisomerase-related gene, while the up-regulated expression of hcp could be justified by the increased secretion of Hcp based on the ELISA test. Overall, this study indicated that CPT has the potential to control the bacterial brown stripe pathogen of rice.

Combined use of flow cytometry and microscopy to study the interactions between the gram-negative betaproteobacterium Acidovorax facilis and uranium(VI).

The former uranium mine Königstein (Saxony, Germany) is currently in the process of remediation by means of controlled underground flooding. Nevertheless, the flooding water has to be cleaned up by a conventional wastewater treatment plant. In this study, the uranium(VI) removal and tolerance mechanisms of the gram-negative betaproteobacterium Acidovorax facilis were investigated by a multidisciplinary approach combining wet chemistry, flow cytometry, and microscopy. The kinetics of uranium removal and the corresponding mechanisms were investigated. The results showed a biphasic process of uranium removal characterized by a first phase where 95% of uranium was removed within the first 8h followed by a second phase that reached equilibrium after 24h. The bacterial cells displayed a total uranium removal capacity of 130mgU/g dry biomass. The removal of uranium was also temperature-dependent, indicating that metabolic activity heavily influenced bacterial interactions with uranium. TEM analyses showed biosorption on the cell surface and intracellular accumulation of uranium. Uranium tolerance tests showed that A. facilis was able to withstand concentrations up to 0.1mM. This work demonstrates that A. facilis is a suitable candidate for in situ bioremediation of flooding water in Königstein as well as for other contaminated waste waters.

The Feasibility of Thermophilic Caldimonas manganoxidans as a Platform for Efficient PHB Production.

Recently, poly(3-hydroxybutyrate) (PHB) has been found in a few thermophilic strains where several advantages can be gained from running fermentation at high temperatures. Caldimonas manganoxidans, a thermophilic gram-negative bacterium, was investigated for the feasibility as a PHB-producing strain. It is suggested that the best fermentation strategy for achieving the highest PHB concentration of 5.4 ± 1.1 g/L (from 20 g/L glucose) in 24 h is to use the fermentation conditions that are favored for the bacterial growth, yet temperature and pH should be chosen at conditions that are favored for the PHB content. Besides, the above fermentation conditions produce PHB that has a high molecular weight of 1274 kDa with a low polydispersity index (PDI) of 1.45, where the highest Mw of PHB of 1399 kDa (PDI of 1.32) is obtained in this study. To the best knowledge of authors, C. manganoxidans has the best PHB productivity among the thermophiles and is comparable to those common PHB-producing mesophiles.

Interspecific competition and protistan grazing affect the coexistence of freshwater betaproteobacterial strains.

The competitive strength of four cosmopolitan freshwater betaproteobacterial isolates was investigated in the presence or absence of bacterivorous flagellates during continuous cultivation in artificial minimal medium at two dilution rates. Bacteria reached similar abundance and growth rate in monocultures, but in co-cultures, two strains (Acidovorax sp. and Massilia sp.) displayed significantly higher numbers and growth rates. These potential cross-feeding benefits were also supported by a high nutritional versatility of the two strains. In contrast, Hydrogenophaga sp. was seemingly less competitive or even inhibited by co-cultivation, and Limnohabitans planktonicus displayed striking abundance fluctuations. The latter two strains were least versatile in the uptake of different carbon sources and thus suffered more from interspecific competition. Moreover, remarkable strain-specific responses appeared when bacteria experienced increasing loss rates due to grazing and/or raised dilution rates. Limnohabitans planktonicus developed no successful defence strategy and was close to extinction. Massilia sp. formed grazing-resistant filaments exclusively at low dilution, but was highly reduced at increased flow-through. Acidovorax sp. was selectively ingested, but compensated grazing losses with accelerated growth rates and formed (co-)aggregates together with Hydrogenophaga sp. to escape predation at high flow-through. These species-specific interactions, growth responses and defence strategies strongly modulate mixed microbial assemblages and the microbial food web.

[Role of the Microbial Community in Formation of Speleothem (Moonmilk) in the Snezhnaya Carst Cave (Abkhazia).]

The resi Its of investigation of speleothem (moonmilk) from the Snezhnaya cave (West Caucasus, Abkhazia) are-reported. The structure of microbial complexes from moonmilk was investigated by quantita- tive PCR; strains of culturable microorganisms were isolated and their preferred temperature and carbon sources were determined. Among eubacteria, -34% belonged to the iron bacteria (Rhodoferax). Most bacte- rial strains were shown to be facultative psychrophiles with the maximum growth rate at 4C. The microstruc- ture and elemental composition of mionmilk were investigated using a scanning electron microscope (EVO- 40HV, Carl Zeiss) and silicon drifi X-ay detector X-MAX 80 ffM2' The visually plastic and homogeneous mass of moonmilk was shown to be highly heterogeneous, containing various microstructures. The elemental composition of some nanostructures depended on the structure of bacterial biofilms. Some loci of the biofilm were found to contain up to 46% (wt/wt) of iron oxides. Calcium content was high, up to 61.5% (wt/wt) only in cubic crystalline structures which were not involved in microbiological processes.

Prey-Specific Growth Responses of Freshwater Flagellate Communities Induced by Morphologically Distinct Bacteria from the Genus Limnohabitans.

Because their large growth potential is counterbalanced with grazing by heterotrophic nanoflagellates (HNF), bacteria of the genus Limnohabitans, which are common in many freshwater habitats, represent a valuable model for examining bacterial carbon flow to the grazer food chain. We conducted experiments with natural HNF communities taken from two distinct habitats, the meso-eutrophic Rímov Reservoir and the oligo-mesotrophic Lake Cep (South Bohemia). HNF communities from each habitat at distinct seasonal phases, a late April algal bloom and a late May clear water phase, were each fed 3 Limnohabitans strains of differing cell sizes. Water samples were prefiltered (5 µm) to release natural HNF communities from zooplankton control and then amended with the Limnohabitans strains L. planktonicus II-D5 (medium sized, rod shaped), Limnohabitans sp. strain T6-5 (thin, long, curved rod), and Limnohabitans sp. strain 2KL-3 (large solenoid). Using temporal sampling and prey treatment, we determined HNF growth parameters such as doubling time, growth efficiency, and length of lag phase prior starting to exponential growth. All three Limnohabitans strains supported HNF growth but in significant prey-, site-, and season-dependent fashions. For instance, addition of the moderately large T6-5 strain yielded very rapid HNF growth with a short lag phase. In contrast, the curved morphology and larger cell size of strain 2KL-3 made this prey somewhat protected against grazing by smaller HNF, resulting in slower HNF growth and longer lag phases. These trends were particularly pronounced during the late May clear-water phase, which was dominated by smaller HNF cells. This may indicate a longer "adaptation time" for the flagellate communities toward the large prey size offered.

Proteomic analysis of nitrate-dependent acetone degradation by Alicycliphilus denitrificans strain BC.

Alicycliphilus denitrificans strain BC grows anaerobically on acetone with nitrate as electron acceptor. Comparative proteomics of cultures of A. denitrificans strain BC grown on either acetone or acetate with nitrate was performed to study the enzymes involved in the acetone degradation pathway. In the proposed acetone degradation pathway, an acetone carboxylase converts acetone to acetoacetate, an AMP-dependent synthetase/ligase converts acetoacetate to acetoacetyl-CoA, and an acetyl-CoA acetyltransferase cleaves acetoacetyl-CoA to two acetyl-CoA. We also found a putative aldehyde dehydrogenase associated with acetone degradation. This enzyme functioned as a ß-hydroxybutyrate dehydrogenase catalyzing the conversion of surplus acetoacetate to ß-hydroxybutyrate that may be converted to the energy and carbon storage compound, poly-ß-hydroxybutyrate. Accordingly, we confirmed the formation of poly-ß-hydroxybutyrate in acetone-grown cells of strain BC. Our findings provide insight in nitrate-dependent acetone degradation that is activated by carboxylation of acetone. This will aid studies of similar pathways found in other microorganisms degrading acetone with nitrate or sulfate as electron acceptor.