Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress.

Agrobacterium tumefaciens GW4 is a heterotrophic arsenite-oxidizing bacterium with a high resistance to arsenic toxicity. It is now a model organism for studying the processes of arsenic detoxification and utilization. Previously, we demonstrated that under low-phosphate conditions, arsenate [As(V)] could enhance bacterial growth and be incorporated into biomolecules, including lipids. While the basic microbial As(V) resistance mechanisms have been characterized, global metabolic responses under low phosphate remain largely unknown. In the present work, the impacts of As(V) and low phosphate on intracellular metabolite and lipid profiles of GW4 were quantified using liquid chromatography-mass spectroscopy (LC-MS) in combination with transcriptional assays and the analysis of intracellular ATP and NADH levels. Metabolite profiling revealed that oxidative stress response pathways were altered and suggested an increase in DNA repair. Changes in metabolite levels in the tricarboxylic acid (TCA) cycle along with increased ATP are consistent with As(V)-enhanced growth of A. tumefaciens GW4. Lipidomics analysis revealed that most glycerophospholipids decreased in abundance when As(V) was available. However, several glycerolipid classes increased, an outcome that is consistent with maximizing growth via a phosphate-sparing phenotype. Differentially regulated lipids included phosphotidylcholine and lysophospholipids, which have not been previously reported in A. tumefaciens The metabolites and lipids identified in this study deepen our understanding of the interplay between phosphate and arsenate on chemical and metabolic levels.IMPORTANCE Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited. Arsenate and phosphate are chemically similar, and this behavior is believed to represent a phosphate-sparing phenotype in which arsenate is used in place of phosphate in certain biomolecules. The research presented here uses a global approach to track metabolic changes in an environmentally relevant bacterium during exposure to arsenate when phosphate is low. Our findings are relevant for understanding the environmental fate of arsenic as well as how human-associated microbiomes respond to this common toxin.

Nocardioides immobilis sp. nov., isolated from iron mine soil.

A Gram-positive, non-motile, rod-shaped aerobic bacterial strain FLL521T was isolated from iron mine soil. Phylogenetic trees based on 16S rRNA gene sequences showed that strain FLL521T belonged to genus Nocardioides in family Nocardioidaceae, with the highest sequence identity to Nocardioides panacisoli GSoil 346T (96.3 %). The DNA G+C content of strain FLL521T was 70.3 mol%. The cell-wall peptidoglycan type was ll-2,6-diaminopimelic acid. The major respiratory quinone was MK-8(H4), and the predominant fatty acids (>5 %) were iso-C16 : 0, C18 : 1ω9c and C17 : 1ω8c. The polar lipids of strain FLL521T were phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and three unidentified phospholipids. Strain FLL521T showed physiological and biochemical differences with the related strains in the Voges-Proskauer test, hydrolysis of l-arginine, utilization of d-mannose, inositol, phenylalanine and acid production from l-arabinose. Based on the phenotypic, genotypic and phylogenetic analyses, strain FLL521T represents a novel species of Nocardioides, for which the name Nocardioides immobilis sp. nov. is proposed. The type strain is FLL521T (=KCTC 39931T=CCTCC AB 2017083T).

Synthesis and biological evaluation of 4-fluoroproline and 4-fluoropyrrolidine-2-acetic acid derivatives as new GABA uptake inhibitors.

Preparation for the N-alkylated derivatives of enantiomerically pure (2S)-4-fluoroproline and (2S)-4-fluoropyrrolidine-2-acetic acid is described. The final compounds were evaluated as potential GAT-1 uptake inhibitors via cultured cell lines expressing mouse GAT-1. Compared with their corresponding 4-hydroxy compounds, these derivatives exhibited slight improvement on their inhibitory potency, but still much weaker than their corresponding compounds with no substituents at the C-4 of the pyrrolidine moiety, with the most potent affinity being about 1/15 fold as that of Tiagabine. The drastic decrease of their affinity may arise from sharp reduction of their basicity due to strong inductive effect of the 4-fluorine. However the configuration of the C-4 linking fluorine did not have much influence on their affinity for GAT-1.

Regulation of Class A ß-Lactamase CzoA by CzoR and IscR in Comamonas testosteroni S44.

A genomic analysis of Comamonas testosteroni S44 revealed a gene that encodes a LysR family transcriptional regulator (here named czoR, czo for cefazolin) located upstream of a putative class A ß-lactamase encoding gene (here named czoA). A putative DNA-binding motif of the Fe-S cluster assembly regulator IscR was identified in the czoR-czoA intergenic region. Real-time RT-PCR and lacZ fusion expression assays indicated that transcription of czoA and czoR were induced by multiple ß-lactams. CzoA expressed in Escherichia coli was shown to contribute to susceptibility to a wide range of ß-lactams judged from minimum inhibitory concentrations. In vitro enzymatic assays showed that CzoA hydrolyzed seven ß-lactams, including benzylpenicillin, ampicillin, cefalexin, cefazolin, cefuroxime, ceftriaxone, and cefepime. Deletion of either iscR or czoR increased susceptibility to cefalexin and cefazolin, while complemented strains restored their wild-type susceptibility levels. Electrophoretic mobility shift assays (EMSA) demonstrated that CzoR and IscR bind to different sites of the czoR-czoA intergenic region. Precise CzoR- and IscR-binding sites were confirmed via DNase I footprinting or short fragment EMSA. When cefalexin or cefazolin was added to cultures, czoR deletion completely inhibited czoA expression but did not affect iscR transcription, while iscR deletion decreased the expressions of both czoR and czoA. These results reveal that CzoR positively affects the expression of czoA with its own expression upregulated by IscR.

Draft genome sequence of Cellulomonas carbonis T26(T) and comparative analysis of six Cellulomonas genomes.

Most Cellulomonas strains are cellulolytic and this feature may be applied in straw degradation and bioremediation. In this study, Cellulomonas carbonis T26(T), Cellulomonas bogoriensis DSM 16987(T) and Cellulomonas cellasea 20108(T) were sequenced. Here we described the draft genomic information of C. carbonis T26(T) and compared it to the related Cellulomonas genomes. Strain T26(T) has a 3,990,666 bp genome size with a G + C content of 73.4 %, containing 3418 protein-coding genes and 59 RNA genes. The results showed good correlation between the genotypes and the physiological phenotypes. The information are useful for the better application of the Cellulomonas strains.

Global Regulator IscR Positively Contributes to Antimonite Resistance and Oxidation in Comamonas testosteroni S44.

Antimonial compounds can be found as a toxic contaminant in the environment. Knowledge on mechanisms of microbial Sb oxidation and its role in microbial tolerance are limited. Previously, we found that Comamonas testosteroni S44 was resistant to multiple heavy metals and was able to oxidize the toxic antimonite [Sb(III)] to the much less toxic antimonate [Sb(V)]. In this study, transposon mutagenesis was performed in C. testosteroni S44 to isolate genes responsible for Sb(III) resistance and oxidation. An insertion mutation into iscR, which regulates genes involved in the biosynthesis of Fe-S clusters, generated a strain called iscR-280. This mutant strain was complemented with a plasmid carrying iscR to generate strain iscR-280C. Compared to the wild type S44 and iscR-280C, strain iscR-280 showed lower resistance to Sb(III) and a lower Sb(III) oxidation rate. Strain iscR-280 also showed lower resistance to As(III), Cd(II), Cu(II), and H2O2. In addition, intracellular γ-glutamylcysteine ligase (γ-GCL) activity and glutathione (GSH) content were decreased in the mutated strain iscR-280. Real-time RT-PCR and lacZ fusion expression assay indicated that transcription of iscR and iscS was induced by Sb(III). Results of electrophoretic mobility shift assay (EMSA) and bacterial one-hybrid (B1H) system demonstrated a positive interaction between IscR and its promoter region. The diverse defective phenotypes and various expression patterns suggest a role for IscR in contributing to multi-metal(loid)s resistance and Sb(III) oxidation via Fe-S cluster biogenesis and oxidative stress protection. Bacterial Sb(III) oxidation is a detoxification reaction.