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Archives of Microbiology (v.193, #10)

Genomics of iron acquisition in the plant pathogen Erwinia amylovora: insights in the biosynthetic pathway of the siderophore desferrioxamine E by Theo H. M. Smits; Brion Duffy (pp. 693-699).

Genomics has clarified the biosynthetic pathway for desferrioxamine E critical for iron acquisition in the enterobacterial fire blight pathogen Erwinia amylovora. Evidence for each of the individual steps and the role of desferrioxamine E biosynthesis in pathogen virulence and cell protection from host defenses is presented. Using comparative genomics, it can be concluded that desferrioxamine biosynthesis is ancestral within the genera Erwinia and Pantoea.

Keywords: Fire blight; Desferrioxamine; Ferrioxamine; Siderophore biosynthesis

Response to osmotic stress and temperature of the fungus Ustilago maydis by Karina Gabriela Salmerón-Santiago; Juan Pablo Pardo; Oscar Flores-Herrera; Guillermo Mendoza-Hernández; Manuel Miranda-Arango; Guadalupe Guerra-Sánchez (pp. 701-709).

Ustilago maydis is a fungal pathogen which is exposed during its life cycle to both abiotic and biotic stresses before and after the infection of maize. To cope with extreme environmental changes, microorganisms usually accumulate the disaccharide trehalose. We have investigated both the accumulation of trehalose and the activity of trehalase during the adaptation of U. maydis haploid cells to thermal, sorbitol, and NaCl stresses. Sorbitol and sodium chloride induced sustained accumulation of trehalose, while a transient increase was observed under heat stress. Sorbitol stressed cells showed higher trehalase activity compared with control cells and to those stressed by NaCl and high temperature. Addition of cycloheximide, a protein synthesis inhibitor, did not affect the trehalose accumulation during the first 15 min, but basal levels of trehalose were reached after the second period of 15 min. The proteomic analysis of the response of U. maydis to temperature, sorbitol, and salt stresses indicated a complex pattern which highlights the change of 18 proteins involved in carbohydrate and amino acid metabolism, protein folding, redox regulation, ion homeostasis, and stress response. We hypothesize that trehalose accumulation during sorbitol stress in U. maydis might be related to the adaptation of this organism during plant infection.

Keywords: Ustilago maydis ; Trehalose accumulation; Heat stress; Osmotic stress; Yeast trehalase

Biofilm formation in Escherichia coli cra mutants is impaired due to down-regulation of curli biosynthesis by Shamlan M. S. Reshamwala; Santosh B. Noronha (pp. 711-722).

Cra is a pleiotropic regulatory protein that controls carbon and energy flux in enteric bacteria. Recent studies have shown that Cra also regulates other cell processes and influences biofilm formation. The purpose of the present study was to investigate the role of Cra in biofilm formation in Escherichia coli. Congo red-binding studies suggested that curli biosynthesis is impaired in cra mutants. Microarray analysis of wild-type and mutant E. coli cultivated in conditions promoting biofilm formation revealed that the curli biosynthesis genes, csgBAC and csgDEFG, are poorly expressed in the mutant, suggesting that transcription of genes required for curli production is regulated by Cra. Four putative Cra-binding sites were identified in the curli intergenic region, which were experimentally validated by performing electromobility shift assays. Site-directed mutagenesis of three Cra-binding sites in the promoter region of the csgDEFG operon suggests that Cra activates transcription of this operon upon binding to operator regions both downstream and upstream of the transcription start site. Based on the Cra-binding sites identified in this and other studies, the Cra consensus sequence is refined.

Keywords: Cra; Curli; Biofilm; Consensus sequence

Salt adaptation in Acinetobacter baylyi: identification and characterization of a secondary glycine betaine transporter by Miriam Sand; Veronique de Berardinis; Ana Mingote; Helena Santos; Stephan Göttig; Volker Müller; Beate Averhoff (pp. 723-730).

Members of the genus Acinetobacter are well known for their metabolic versatility that allows them to adapt to different ecological niches. Here, we have addressed how the model strain Acinetobacter baylyi copes with different salinities and low water activities. A. baylyi tolerates up to 900 mM sodium salts and even higher concentrations of potassium chloride. Growth at high salinities was better in complex than in mineral medium and addition of glycine betaine stimulated growth at high salinities in mineral medium. Cells grown at high salinities took up glycine betaine from the medium. Uptake of glycine betaine was energy dependent and dependent on a salinity gradient across the membrane. Inspection of the genome sequence revealed two potential candidates for glycine betaine transport, both encoding potential secondary transporters, one of the major facilitator superfamily (MFS) class (ACIAD2280) and one of the betaine/choline/carnitine transporter (BCCT) family (ACIAD3460). The latter is essential for glycine betaine transport in A. baylyi. The broad distribution of ACIAD3460 homologues indicates the essential role of secondary transporters in the adaptation of Acinetobacter species to osmotic stress.

Keywords: Acinetobacter ; Salt adaptation; Glycine betaine transport

Kinetic characterisation of recombinant Corynebacterium glutamicum NAD⁺-dependent LDH over-expressed in E. coli and its rescue of an lldD ⁻ phenotype in C. glutamicum: the issue of reversibility re-examined by Michael A. Sharkey; Marcus A. Maher; Armel Guyonvarch; Paul C. Engel (pp. 731-740).

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD⁺-dependent l-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d-fructose-1,6-bisphosphate (Fru-1,6-P₂). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V _max for the reduction of pyruvate being approximately tenfold that of the value for l-lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P₂, while a sigmoidal response (indicative of positive cooperativity) was seen towards l-lactate without Fru-1,6-P₂. Strikingly, when introduced into an lldD ⁻ strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on l-lactate as the sole carbon source.

Keywords: Enzyme kinetics; Corynebacterium glutamicum ; Lactate dehydrogenase; Unidirectional reaction; Genetic complementation

Characterization of the role of DR0171 in transcriptional response to radiation in the extremely radioresistant bacterium Deinococcus radiodurans by Huiming Lu; Wenrong Xia; Huan Chen; Longfei Yin; Xiaojun Zhao; Guangzhi Xu; Yuejin Hua (pp. 741-750).

The extremely radioresistant bacterium Deinococcus radiodurans encodes a number of function-unknown genes, and some of them involve in the radioresistance. The radiation-inducible gene dr0171 has a recA-like expression pattern in the postirradiation recovery and was also supposed to encode a transcriptional regulator to contribute to the radioresistance. Here, we found that the EGFP-tagged DR0171 proteins gathered in the nucleoid regions after radiation. Further, we constructed a null mutant of dr0171 and found that the incapacitation of the dr0171 led to a significant decline in resistance to γ-rays, UV radiation, and hydrogen peroxide and delayed genomic DNA reconstruction after radiation, indicating that this gene is involved in the postirradiation recovery. The microarray assays showed that the disruption of dr0171 does not lead to a significant change in the transcriptional profile of D. radiodurans under normal conditions, except after the stress of 6 kGy γ radiation was applied. The transcript level of at least 153 genes decreased over twofold in the irradiated dr0171 mutant compared with those in the irradiated wild-type strain, indicating that DR0171 only functions after radiation damage. Taken together, the data presented here indicate that DR0171 serves as a regulator of the transcriptional response to radiation damage in D. radiodurans.

Keywords: Deinococcus radiodurans ; DR0171; Radiation resistance; Microarray; Response regulator

Kinetic properties of Mycobacterium tuberculosis bifunctional GlmU by Yan Zhou; Yi Xin; Shanshan Sha; Yufang Ma (pp. 751-757).

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.

Keywords: M. tuberculosis ; GlmU; Glucosamine-1-phosphate acetyltransferase; N-acetyl-glucosamine-1-phosphate uridyltransferase; Kinetics of GlmU

EmmdR, a new member of the MATE family of multidrug transporters, extrudes quinolones from Enterobacter cloacae by Gui-Xin He; Conner Thorpe; Dennis Walsh; Robert Crow; Huizhong Chen; Sanath Kumar; Manuel F. Varela (pp. 759-765).

We cloned a gene, ECL_03329, from the chromosome of Enterobacter cloacae ATCC13047, using a drug-hypersensitive Escherichia coli KAM32 cell as the host. We show here that this gene, designated as emmdR, is responsible for multidrug resistance in E. cloacae. E. coli KAM32 host cells containing the cloned emmdR gene (KAM32/pEMMDR28) showed decreased susceptibilities to benzalkonium chloride, norfloxacin, ciprofloxacin, levofloxacin, ethidium bromide, acriflavine, rhodamine6G, and trimethoprim. emmdR-deficient E. cloacae cells (EcΔemmdR) showed increased susceptibilities to several of the antimicrobial agents tested. EmmdR has twelve predicted transmembrane segments and some shared identity with members of the multidrug and toxic compound extrusion (MATE) family of transporters. Study of the antimicrobial agent efflux activities revealed that EmmdR is an H⁺-drug antiporter but not a Na⁺ driven efflux pump. These results indicate that EmmdR is responsible for multidrug resistance and pumps out quinolones from E. cloacae.

Keywords: Multidrug transporter; Enterobacter cloacae ; Resistance; MATE Family

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Archives of Microbiology (v.193, #10)

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Archives of Microbiology (v.193, #10)