Inactivation of uptake hydrogenase leads to enhanced and sustained hydrogen production with high nitrogenase activity under high light exposure in the cyanobacterium <Emphasis Type="Italic">Anabaena siamensis</Emphasis> TISTR 8012

Background

Biohydrogen from cyanobacteria has attracted public interest due to its potential as a renewable energy carrier produced from solar energy and water. Anabaena siamensis TISTR 8012, a novel strain isolated from rice paddy field in Thailand, has been identified as a promising cyanobacterial strain for use as a high-yield hydrogen producer attributed to the activities of two enzymes, nitrogenase and bidirectional hydrogenase. One main obstacle for high hydrogen production by A. siamensis is a light-driven hydrogen consumption catalyzed by the uptake hydrogenase. To overcome this and in order to enhance the potential for nitrogenase based hydrogen production, we engineered a hydrogen uptake deficient strain by interrupting hupS encoding the small subunit of the uptake hydrogenase.

Results

An engineered strain lacking a functional uptake hydrogenase (?hupS) produced about 4-folds more hydrogen than the wild type strain. Moreover, the ?hupS strain showed long term, sustained hydrogen production under light exposure with 2–3 folds higher nitrogenase activity compared to the wild type. In addition, HupS inactivation had no major effects on cell growth and heterocyst differentiation. Gene expression analysis using RT-PCR indicates that electrons and ATP molecules required for hydrogen production in the ?hupS strain may be obtained from the electron transport chain associated with the photosynthetic oxidation of water in the vegetative cells. The ?hupS strain was found to compete well with the wild type up to 50 h in a mixed culture, thereafter the wild type started to grow on the relative expense of the ?hupS strain.

Conclusions

Inactivation of hupS is an effective strategy for improving biohydrogen production, in rates and specifically in total yield, in nitrogen-fixing cultures of the cyanobacterium Anabaena siamensis TISTR 8012.

     

The effects of gibberellins and mepiquat chloride on nitrogenase activity in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Application of plant growth regulators (PGRs) to soybean plants is known to induce changes in nitrogenase activity in root nodules, and this led us to hypothesize that PGRs would affect nitrogenase activity in free-living rhizobia cultures. Little is known about the molecular basis of the effects of PGRs on nitrogenase activity in free-living rhizobia cultures. Therefore, a comparative study was conducted on the effects of gibberellins (GA₃) and mepiquat chloride (PIX), which regulate plant growth, on the nitrogenase activity of the nitrogen-fixing bacterium Bradyrhizobium japonicum. Fix and nif gene regulation and protein expression in free-living cultures of B. japonicum were investigated using real-time PCR and two-dimensional electrophoresis after treatment with GA₃ or PIX. GA₃ treatment decreased nitrogenase activity and the relative expression of nifA, nifH, and fixA genes, but these effects were reversed by PIX treatment. As expected, several proteins involved in nitrogenase synthesis were down-regulated in the GA₃-treated group. Conversely, several proteins involved in nitrogenase synthesis were up-regulated in the PIX-treated group, including bifunctional ornithine acetyltransferase/N-acetylglutamate synthase, transaldolase, ubiquinol-cytochrome C reductase iron-sulfur subunit, electron transfer flavoprotein subunit beta, and acyl-CoA dehydrogenase. Two-pot experiments were conducted to evaluate the effects of GA₃ and PIX on nodulation and nitrogenase activity in Rhizobium-treated legumes. Interestingly, GA₃ treatment increased nodulation and depressed nitrogenase activity, but PIX treatment decreased nodulation and enhanced nitrogenase activity. Our data show that the nif and fix genes, as well as several proteins involved in nitrogenase synthesis, are up-regulated by PIX and down-regulated by GA₃, respectively, in B. japonicum.     

A new bacterial strain, <Emphasis Type="Italic">Pseudomonas koreensis</Emphasis> IB-4, as a promising agent for plant pathogen biological control

A bacterial strain IB-4, antagonistic to plant pathogenic fungi of the genera Fusarium, Bipolaris, and Alternaria, was isolated from arable soils of the Mechetlinskii district, Bashkortostan. Physiological, biochemical, and culture morphological properties of strain IB-4 supported its classification within the genus Pseudomonas. In spite of some discrepancies in the results of phenotypic and chemotaxonomic research, analysis of the 16S rRNA gene sequence, DNA–DNA hybridization, GC-content, and MALDI mass spectral data provide considerable evidence supporting its identification as a Pseudomonas koreensis strain. P. koreensis strain IB-4 was shown to possess the valuable features characteristic of PGPR microorganisms: antifungal and nitrogenase activities and ability to synthesize indole-3-acetic acid (IAA) and cytokinin-like compounds. Field test, in which potato plants were treated with the culture liquid of P. koreensis strain IB-4 revealed a positive effect on potato yield and resistance to plant pathogens.     

Genealogical analysis of the use of two wheatgrass (<Emphasis Type="Italic">Agropyron</Emphasis>) species in common wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding for disease resistance

During the last 80 years, in order to increase the genetic variability of wheat, translocations containing nine elongated wheatgrass (Agropyron elongatum) and eight intermediate wheatgrass (Agropyron intermedium) genes, which control resistance to pathogens, were transferred to this crop culture. Genealogical and statistical analysis of 1500 varieties developed using the wheatgrass gave evidence of the continuing increase in the proportion of such varieties in the total number of wheat varieties over the last half-century. Translocations from Ag. elongatum most commonly occur in the pedigrees of the varieties from the United States, less frequently they can be found in Australian and Chinese varieties, and they are extremely rare—in European and African ones. Ag. intermedium most frequently occurs in the pedigrees of the Eastern European varieties, mainly in those from Russia, as well as in the varieties from China. The observed uneven distribution of such varieties may be associated with either the effectiveness of the translocation in the development of resistance to the local populations of pathogens or with the effect of the translocation on the adaptive traits of plants. By computer tracking of pedigrees, we performed an inventory of the translocation donors from Ag. elongatum and Ag. intermedium used in the breeding programs in the United States, Russia, Australia, India, and China. The most widely occurring combinations of the gene complex Lr24/Sr24 of Ag. elongatum with other resistance genes were revealed. In Russia, there were developed varieties in which the 6D chromosome was substituted by the 6Ai chromosome of Ag. intermedium, which controls disease resistance and the adaptivity of plants. The identification and introgression of new translocations indicates that the possibilities of using wheatgrass species for broadening of genetic variability of wheat are far from being exhausted.     

Dual-species biofilm of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> on stainless steel surface

Listeria monocytogenes is a Gram-positive bacterium commonly associated with foodborne diseases. Due its ability to survive under adverse environmental conditions and to form biofilm, this bacterium is a major concern for the food industry, since it can compromise sanitation procedures and increase the risk of post-processing contamination. Little is known about the interaction between L. monocytogenes and Gram-negative bacteria on biofilm formation. Thus, in order to evaluate this interaction, Escherichia coli and L. monocytogenes were tested for their ability to form biofilms together or in monoculture. We also aimed to evaluate the ability of L. monocytogenes 1/2a and its isogenic mutant strain (ΔprfA ΔsigB) to form biofilm in the presence of E. coli. We assessed the importance of the virulence regulators, PrfA and σ^B, in this process since they are involved in many aspects of L. monocytogenes pathogenicity. Biofilm formation was assessed using stainless steel AISI 304 #4 slides immersed into brain heart infusion broth, reconstituted powder milk and E. coli preconditioned medium at 25 °C. Our results indicated that a higher amount of biofilm was formed by the wild type strain of L. monocytogenes than by its isogenic mutant, indicating that prfA and sigB are important for biofilm development, especially maturation under our experimental conditions. The presence of E. coli or its metabolites in preconditioned medium did not influence biofilm formation by L. monocytogenes. Our results confirm the possibility of concomitant biofilm formation by L. monocytogenes and E. coli, two bacteria of major significance in the food industry.     

The eurythermal adaptivity and temperature tolerance of a newly isolated psychrotolerant Arctic <Emphasis Type="Italic">Chlorella</Emphasis> sp.

A new strain of Chlorella sp. (Chlorella-Arc), isolated from Arctic glacier melt water, was found to have high specific growth rates (μ) between 3 and 27 °C, with a maximum specific growth rate of 0.85 day^?1 at 15 °C, indicating that this strain was a eurythermal strain with a broad temperature tolerance range. To understand its acclimation strategies to low and high temperatures, the physiological and biochemical responses of the Chlorella-Arc to temperature were studied and compared with those of a temperate Chlorella pyrenoidosa strain (Chlorella-Temp). As indicated by declining F _v/F _m, photoinhibition occurred in Chlorella-Arc at low temperature. However, Chlorella-Arc reduced the size of the light-harvesting complex (LHC) to alleviate photoinhibition, as indicated by an increasing Chl a/b ratio with decreasing temperatures. Interestingly, Chlorella-Arc tended to secrete soluble sugar into the culture medium with increasing temperature, while its intracellular soluble sugar content did not vary with temperature changes, indicating that the algal cells might suffer from osmotic stress at high temperature, which could be adjusted by excretion of soluble sugar. Chlorella-Arc accumulated protein and lipids under lower temperatures (<15 °C), and its metabolism switched to synthesis of soluble sugar as temperatures rose. This reflects a flexible ability of Chlorella-Arc to regulate carbon and energy distribution when exposed to wide temperature shifts. More saturated fatty acids (SFA) in Chlorella-Arc than Chlorella-Temp also might serve as the energy source for growth in the cold and contribute to its cold tolerance.     

Ochrobactrum intermedium ANKI, a nitrogen-fixing bacterium able to decolorize azobenzene

Morphological and biochemical properties of the nitrogen-fixing strain Ochrobactrum intermedium ANKI, intensely growing on media with azo compounds, and its resistance to various common xenobiotics were investigated. The kinetics of azobenzene conversion by O. intermedium ANKI was studied. Under cometabolism conditions, up to 40 mg of azobenzene per liter of medium were decolorized within one week. It was shown that the strain possessed molybdenum-dependent nitrogenase activity, and its nitrogenase system was sensitive to oxygen and fixed nitrogen in the medium.     

In silico characterization and transcriptomic analysis of nif family genes from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

In silico approaches in conjunction with morphology, nitrogenase activity, and qRT-PCR explore the impact of selected abiotic stressor such as arsenic, salt, cadmium, copper, and butachlor on nitrogen fixing (nif family) genes of diazotrophic cyanobacterium Anabaena sp. PCC7120. A total of 19 nif genes are present within the Anabaena genome that is involved in the process of nitrogen fixation. Docking studies revealed the interaction between these nif gene-encoded proteins and the selected abiotic stressors which were further validated through decreased heterocyst frequency, fragmentation of filaments, and downregulation of nitrogenase activity under these stresses indicating towards their toxic impact on nitrogen fixation potential of filamentous cyanobacterium Anabaena sp. PCC7120. Another appealing finding of this study is even though having similar binding energy and similar interacting residues between arsenic/salt and copper/cadmium to nif-encoded proteins, arsenic and cadmium are more toxic than salt and copper for nitrogenase activity of Anabaena which is crucial for growth and yield of rice paddy and soil reclamation.     

Functional analysis of alcohol dehydrogenase (<Emphasis Type="Italic">ADH</Emphasis>) genes in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To characterize the genes responsible for ethanol utilization in Pichia pastoris.

Results

ADH3 (XM_002491337) and ADH (FN392323) genes were disrupted in P. pastoris. The ADH3 mutant strain, MK115 (Δadh3), lost its ability to grow on minimal ethanol media but produced ethanol in minimal glucose medium. ADH3p was responsible for 92 % of total Adh enzyme activity in glucose media. The double knockout strain MK117 (Δadh3Δadh) also produced ethanol. The Adh activities of X33 and MK116 (Δadh) strains were not different. Thus, the ADH gene does not play a role in ethanol metabolism.

Conclusion

The PpADH3 is the only gene responsible for consumption of ethanol in P. pastoris.

     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

Biosynthesis of poly(2-hydroxybutyrate-co-lactate) in metabolically engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

We have previously reported in vivo biosynthesis of polyhydroxyalkanoates containing 2-hydroxyacid monomers such as lactate and 2-hydroxybutyrate in recombinant Escherichia coli strains by the expression of evolved Clostridium propionicum propionyl-CoA transferase (PctCp) and Pseudomonas sp. MBEL 6-19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 _Ps6-19). Here, we report the biosynthesis of poly(2-hydroxybutyrate-co-lactate)[P(2HB-co-LA)] by direct fermentation of metabolically engineered E. coli strain. Among E. coli strains WL3110, XL1-Blue, and BL21(DE3), recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 produced P(76.4mol%2HB-co-23.6mol%LA) to the highest content of 88 wt% when it was cultured in a chemically defined medium containing 20 g/L of glucose and 2 g/L of sodium 2-hydroxybutyrate. When recombinant E. coli XL1-Blue strain expressing PhaC1437 and Pct540 was cultured in a chemically defined medium containing 20 g/L of glucose and varying concentration of sodium 2-hydroxybutyrate, 2HB monomer fraction in P(2HB-co-LA) increased proportional to the concentration of sodium 2-hydroxybutyrate added to the culture medium. P(2HB-co-LA)] could also be produced from glucose as a sole carbon source without sodium 2-hydroxybutyrate into the culture medium. Recombinant E. coli XL1-Blue strain expressing the phaC1437, pct540, cimA3.7, and leuBCD genes together with the L. lactis Il1403 panE gene, successfully produced P(23.5mol%2HB-co-76.5mol%LA)] to the polymer content of 19.4 wt% when it cultured in a chemically defined medium containing 20 g/L of glucose. The metabolic engineering strategy reported here should be useful for the production of novel copolymer P(2HB-co-LA)].     

Intermicrobial relationships of the pea nodule symbiont <Emphasis Type="Italic">Serratia</Emphasis> sp. Ent16 and its colonization of the host endorhizosphere

A strain of Serratia sp. Ent16 isolated from internal tissues of pea nodule inhibited in vitro growth of the plant pathogens Fusarium oxysporum and Bipolaris sorokiniana and the model strain Rhizobium leguminosarum bv viceae 1078 but had a considerably weaker antagonistic effect on the Rhizobium strain Rh16 from its own nodule. Cells of the Ent16 strain tagged by the gfp gene (the Ent16-gfp strain) were not seen in the pea endorhizosphere when plants were grown in a rich culture medium. The development of symbiosis was favored by plant germination on filter paper. Confocal microscopy showed that individual cells of the Ent16-gfp strain were attached to the outer side of root hair cell walls, while agglomerations of fluorescent bacterial cells were detected in the zone of exoderm of lateral root formation and in root vessels. A series of scanned sections of pea root revealed the presence of the Ent16-gfp strain in lateral root primordia, through which the bacteria penetrated the endorhizosphere.     

Biotechnological potential of a rhizosphere <Emphasis Type="Italic">Pseudomonas</Emphasis><Emphasis Type="Italic">aeruginosa</Emphasis> strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide

Bacterial phenazine metabolites belong to a group of nitrogen-containing heterocyclic compounds with antimicrobial activities. In this study, a rhizosphere Pseudomonas aeruginosa strain PA1201 was isolated and identified through 16S rDNA sequence analysis and fatty acid profiling. PA1201 inhibited the growth of various pathogenic microorganisms, including Rhizotonia solani, Magnaporthe grisea, Fusarium graminearum, Xanthomonas oryzae pv. oryzae, Xanthomonas oryzae pv. oryzicola, and Staphylococcus aureus. High Performance Liquid Chromatography showed that PA1201 produced high levels of phenazine-1-carboxylic acid (PCA), a registered green fungicide ‘Shenqinmycin’ with the fermentation titers of 81.7 mg/L in pigment producing medium (PPM) and 926.9 mg/L in SCG medium containing soybean meal, corn steep liquor and glucose. In addition, PA1201 produced another antifungal metabolite, phenazine-1-carboxaminde (PCN), a derivative of PCA, with the fermentation titers of 18.1 and 489.5 mg/L in PPM and SCG medium respectively. To the best of our knowledge, PA1201 is a rhizosphere originating P. aeruginosa strain that congenitally produces the highest levels of PCA and PCN among currently reported P. aeruginosa isolates, which endows it great biotechnological potential to be transformed to a biopesticide-producing engineering strain.     

Identification of alcohol stress tolerance genes of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 using adaptive laboratory evolution

Background

Synechocystis sp. PCC 6803 is an attractive organism for the production of alcohols, such as isobutanol and ethanol. However, because stress against the produced alcohol is a major barrier for industrial applications, it is highly desirable to engineer organisms with strong alcohol tolerance.

Results

Isobutanol-tolerant strains of Synechocystis sp. PCC 6803 were obtained by long-term passage culture experiments using medium containing 2 g/L isobutanol. These evolved strains grew on medium containing 5 g/L isobutanol on which the parental strain could not grow. Mutation analysis of the evolved strains revealed that they acquired resistance ability due to combinatorial malfunctions of slr1044 (mcpA) and slr0369 (envD), or slr0322 (hik43) and envD. The tolerant strains demonstrated stress resistance against isobutanol as well as a wide variety of alcohols such as ethanol, n-butanol, and isopentanol. As a result of introducing an ethanol-producing pathway into the evolved strain, its productivity successfully increased to 142% of the control strain.

Conclusions

Novel mutations were identified that improved the stress tolerance ability of various alcohols in Synechocystis sp. PCC 6803.

     

Growth and wax ester production of an <Emphasis Type="Italic">Acinetobacter baylyi</Emphasis> ADP1 mutant deficient in exopolysaccharide capsule synthesis

Acinetobacter baylyi ADP1 naturally produces wax esters that could be used as a raw material in industrial applications. We attempted to improve wax ester yield of A. baylyi ADP1 by removing rmlA, a gene involved in exopolysaccharide production. Growth rate, biomass formation and wax ester yield on 4-hydroxybenzoate were not affected, but the rmlA ^? strain grew slower on acetate, while reaching similar biomass and wax ester yield. The rmlA ^? cells had malformed shape and large size and grew poorly on glucose without expression of the gene for pyruvate kinase (pykF) from Escherichia coli. The pykF-expressing rmlA ^? strain had similar growth rate, lowered biomass formation and improved wax ester production on glucose as compared to the wild-type strain expressing pykF. Cultivation of the pykF-expressing rmlA ^? strain on an elevated glucose concentration in a medium supplemented with amino acids resulted in doubled molar wax ester yield and acetate production.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of endangered <Emphasis Type="Italic">Mammillaria</Emphasis> genus (Cactaceae) species and genetic stability assessment using SSR markers

In vitro propagation protocols were established for endangered species of cacti Mammillaria hernandezii, M. dixanthocentron, and M. lanata. In vitro-germinated seedlings were used as the explant source. Three explant types were evaluated as apical, basal, and lateral stem sections. Shoot multiplication was achieved using Murashige and Skoog (MS) medium supplemented with benzyladenine, kinetin, meta-topolin, and thidiazuron in equimolar concentrations (0.0, 0.4, 1.1, 2.2, 4.4, and 8.9 μM). Shoot regeneration was obtained primarily in the lateral stem section explants. In M. hernandezii, an average of 7.4 shoots was regenerated in MS medium with 2.2 μM meta-topolin. M. dixanthocentron and M. lanata averaged 16.7 and 17.9 shoots/explant, respectively, in MS medium supplemented with 1.1 μM meta-topolin. Rooting occurred in MS medium without growth regulators. Three in vitro culture cycles were performed to validate the propagation protocols and to verify genetic stability. Shoots were collected in each cycle and genomic DNA was extracted. Amplified microsatellites were used to compare each genotype with its respective donor plant. Polymorphic information content analysis showed low levels of intra-clonal polymorphisms—M. hernandezii 0.04 and M. dixanthocentron and M. lanata both 0.12. More than 95% of the plants were successfully acclimatized in the greenhouse. After 12 months, plants of M. hernandezii reached the flowering stage; M. dixanthocentron and M. lanata flowered at 24 mo.