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Archives of Microbiology (v.193, #7)

Identification and functional analysis of a nitrate assimilation operon nasACKBDEF from Amycolatopsis mediterranei U32 by Zhihui Shao; Jin Gao; Xiaoming Ding; Jin Wang; Juishen Chiao; Guoping Zhao (pp. 463-477).

Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the “nitrate stimulated” rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.

Keywords: Amycolatopsis mediterranei ; Nitrate assimilation; nas operon; Rifamycin

Characterization of long-chain fatty acid uptake in Caulobacter crescentus by Fred Zalatan; Paul Black (pp. 479-487).

Studies evaluating the uptake of long-chain fatty acids in Caulobacter crescentus are consistent with a protein-mediated process. Using oleic acid (C18:1) as a substrate, fatty acid uptake was linear for up to 15 min. This process was saturable giving apparent V_max and K_m values of 374 pmol oleate transported/min/mg total protein and 61 μM oleate, respectively, consistent with the notion that one or more proteins are likely involved. The rates of fatty acid uptake in C. crescentus were comparable to those defined in Escherichia coli. Uncoupling the electron transport chain inhibited oleic acid uptake, indicating that like the long-chain fatty acid uptake systems defined in other gram-negative bacteria, this process is energy-dependent in C. crescentus. Long-chain acyl CoA synthetase activities were also evaluated to address whether vectorial acylation represented a likely mechanism driving fatty acid uptake in C. crescentus. These gram-negative bacteria have considerable long-chain acyl CoA synthetase activity (940 pmol oleoyl CoA formed/min/mg total protein), consistent with the notion that the formation of acyl CoA is coincident with uptake. These results suggest that long-chain fatty acid uptake in C. crescentus proceeds through a mechanism that is likely to involve one or more proteins.

Keywords: Fatty acid transport; Acyl CoA synthetase; Gram-negative bacteria; Stalked bacteria; Differentiating bacteria

Plant growth promotion traits of phosphobacteria isolated from Puna, Argentina by Emilce Viruel; María E. Lucca; Faustino Siñeriz (pp. 489-496).

The ability of soil microorganisms to solubilize phosphate is an important trait of plant growth-promoting bacteria leading to increased yields and smaller use of fertilizers. This study presents the isolation and characterization of phosphobacteria from Puna, northwestern Argentina and the ability to produce phosphate solubilization, alkaline phosphatase, siderophores, and indole acetic acid. The P-solubilizing activity was coincidental with a decrease in pH values of the tricalcium phosphate medium for all strains after 72 h of incubation. All the isolates showed the capacity to produce siderophores and indoles. Identification by 16S rDNA sequencing and phylogenetic analysis revealed that these strains belong to the genera Pantoea, Serratia, Enterobacter, and Pseudomonas. These isolates appear attractive for exploring their plant growth-promoting activity and potential field application.

Keywords: Phosphobacteria; Phosphate solubilization; Indol acetic acid; Siderophore; 16S rDNA

Alleviation of cold stress in inoculated wheat (Triticum aestivum L.) seedlings with psychrotolerant Pseudomonads from NW Himalayas by Pankaj Kumar Mishra; Shekhar Chandra Bisht; Pooja Ruwari; Govindan Selvakumar; Gopal Krishna Joshi; Jaideep Kumar Bisht; Jagdish Chandra Bhatt; Hari Shankar Gupta (pp. 497-513).

Twelve psychrotolerant Pseudomonad strains were selected on the basis of various plant growth-promoting (PGP) activities at cold temperature (4°C). The effect of inoculation with Pseudomonad strains on cold alleviation and growth of wheat seedling at cold temperature (8°C) was investigated under greenhouse condition. Inoculation with Pseudomonad strains significantly enhanced root/shoot biomass and nutrients uptake as compared to non-bacterized control at 60 days of plant growth. Bacterization significantly improved the level of cellular metabolites like chlorophyll, anthocyanin, free proline, total phenolics, starch content, physiologically available iron, proteins, and amino acids that are sign of alleviation of cold stress in wheat plants. Increased relative water content, reduced membrane injury (electrolyte leakage), and Na⁺/K⁺ ratio were also recorded in bacterized wheat plants. Electrolyte leakage and Na⁺/K⁺ were found inversely proportional to plant growth at cold temperature. Statistical analysis of twenty-three measured parameters revealed that uninoculated control was under cold stress while eight bacterial strains were positively alleviating cold stress in wheat plants. Thus, the psychrotrophic Pseudomonad strains could effectively provide a promising solution to overcome cold stress, which is major factor hindering wheat productivity under cold climatic condition.

Keywords: Cold alleviation; Wheat; Psychrotrophic Pseudomonad; PGPR; NW Himalayas

The Saccharomyces cerevisiae flavodoxin-like proteins Ycp4 and Rfs1 play a role in stress response and in the regulation of genes related to metabolism by Fernando Cardona; Helena Orozco; Sylvie Friant; Agustín Aranda; Marcel·lí del Olmo (pp. 515-525).

SPI1 is a gene whose expression responds to many environmental stimuli, including entry into stationary phase. We have performed a screening to identify genes that activate SPI1 promoter when overexpressed. The phosphatidylinositol-4-phosphate 5-kinase gene MSS4 was identified as a positive activator of SPI1. Another SPI1 transcriptional regulator isolated was the flavodoxin-like gene YCP4. YCP4 and its homolog RFS1 regulate the expression of many genes during the late stages of growth. The double deletion mutant in YCP4 and its homolog RFS1 has an impact on gene expression related to metabolism by increasing the expression of genes involved in hexose transport and glycolysis, and decreasing expression of genes of amino acid metabolism pathways. Genes related to mating and response to pheromone show a decreased expression in the double mutant, while transcription of genes involved in translational elongation is increased. Deletion of these genes, together with the third member of the family, PST2, has a complex effect on the stress response. For instance, double mutant ycp4Δrfs1Δ has an increased response to oxidative stress, but a decreased tolerance to cell-damaging agent SDS. Additionally, this mutation affects chronological aging and slightly increases fermentative capacity.

Keywords: S. cerevisiae ; Stress; Flavodoxin-like; YCP4 ; RFS1

Endophytic bacteria of Mammillaria fraileana, an endemic rock-colonizing cactus of the southern Sonoran Desert by Blanca R. Lopez; Yoav Bashan; Macario Bacilio (pp. 527-541).

The small cactus Mammillaria fraileana is a pioneer rock-colonizing plant harboring endophytic bacteria with the potential for nitrogen fixation and rock weathering (phosphate solubilization and rock degradation). In seeds, only a combination of culture-independent methods, such as fluorescence in situ hybridization, scanning electron microscopy, and fluorescence vital staining, detected significant amounts of non-culturable, but living, endophytic bacteria distributed underneath the membrane covering the embryo, in the undifferentiated tissue of the embryo, and in the vascular tissue. Large populations of culturable endophytic bacteria were detected in stems and roots of wild plants colonizing rocks in the southern Sonoran Desert, but not in seeds. Among 14 endophytic bacterial isolates found in roots, four isolates were identified by full sequencing of their 16S rRNA gene. In vitro tests indicated that Azotobacter vinelandii M2Per is a potent nitrogen fixer. Solubilization of inorganic phosphate was exhibited by Pseudomonas putida M5TSA, Enterobacter sakazakii M2PFe, and Bacillus megaterium M1PCa, while A. vinelandii M2Per, P. putida M5TSA, and B. megaterium M1PCa weathered rock by reducing the size of rock particles, probably by changing the pH of the liquid media. Cultivated seedlings of M. fraileana, derived from disinfected seeds and inoculated with endophytic bacteria, showed re-colonization 105 days after inoculation. Their densities decreased from the root toward the stem and apical zones. Functional traits in planta of culturable and non-culturable endophytic bacteria in seeds remain unknown.

Keywords: Cactus; Plant colonization of rocks; Endophytic bacteria; Mammillaria ; Phosphate solubilization; Rock weathering

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Archives of Microbiology (v.193, #7)

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Archives of Microbiology (v.193, #7)