Pyomelanin is a brown/black extracellular pigment with antioxidant and iron acquisition properties that is produced by a number of different bacteria. Production of pyomelanin in Pseudomonas aeruginosa contributes to increased resistance to oxidative stress and persistence in chronic infections. We demonstrate that pyomelanin production can be inhibited by 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC). This treatment increases sensitivity of pyomelanogenic P. aeruginosa strains to oxidative stress, without altering the growth rate or resistance to aminoglycosides. As such, NTBC has potential to function as an anti-virulence factor in treating pyomelanogenic bacterial infections.Electronic supplementary materialThe online version of this article (doi:10.1007/s00284-014-0593-9) contains supplementary material, which is available to authorized users.

A psychrophilic, Gram-negative bacterium, designated MOL-1 T , was isolated from water of Lake . The major cellular fatty acids were iso-C 15 : 0 , iso-C 17 : 0 3-OH and summed feature 3 (iso-C 15 : 0 2-OH and/or C 16 : 1 v7c). Menaquinone MK-7 is the predominant respiratory quinone, while symhomospermidine is the predominant polyamine. The polar lipid profile is composed of the predominant lipids phosphatidylethanolamine and unidentified polar lipid L2, with moderate amounts of unidentified polar lipids L1, L5 and L6 and unidentified aminophospholipids APL1 and APL2 and minor to trace amounts of unidentified polar lipids L3, L4, L7, L8, L9 and L10, unidentified phospholipid PL4 and unidentified aminophospholipid APL3. After molecular and phenotypic studies, including chemotaxonomic analyses, it was concluded that strain MOL-1 T represents a novel Sphingobacterium species, for which the name Sphingobacterium psychroaquaticum sp. nov. is proposed. The type strain is MOL-1 T (5NRRL B-59232 T 5DSM T).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Pyomelanin is a reddish-brown pigment that provides bacteria and fungi protection from oxidative stress, and is reported to contribute to infection persistence. Production of this pigment can be inhibited by the anti-virulence agent 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC). The Pseudomonas aeruginosa clinical isolate DKN343 exhibited high levels of resistance to NTBC, and the mechanism of pyomelanin production in this strain was uncharacterized. We determined that pyomelanin production in the clinical Pseudomonas aeruginosa isolate DKN343 was due to a loss of function in homogentisate 1,2-dioxygenase (HmgA). Several potential resistance mechanisms were investigated, and the MexAB-OprM efflux pump is required for resistance to NTBC. DKN343 has a frameshift mutation in NalC, which is a known indirect repressor of the mexAB-oprM operon. This frameshift mutation may contribute to the increased resistance of DKN343 to NTBC. Additional studies investigating the prevalence of resistance in pyomelanogenic microbes are necessary to determine the future applications of NTBC as an anti-virulence therapy.

Rhizobium sp. IRBG74 (IRBG74), is a symbiont of Sesbania cannabina and a growth promoting endophyte of rice. Here, we compare the transcriptomic and physiological responses of IRBG74 in the rhizosphere of S. cannabina and rice. MethodsWe used RNA sequencing to determine transcriptomic changes at 12 and 72 hours post inoculation (hpi) in rhizosphere. Upregulation of key pathways was con rmed using β-glucuronidase (GUS) reporter strains and by histochemical and quantitative GUS activity. ResultsSigni cant changes in transcriptome with S. cannabina were detected at 12hpi but most gene expression changes with rice were observed at 72hpi. Many pathways including Nod factor synthesis, two component systems, ABC transporters, and synthesis of indole acetic acid (IAA) were upregulated whereas translation, RNA degradation, protein export and sulfur metabolism were downregulated with both plants. In contrast, motility and chemotaxis genes were induced speci cally with S. cannabina. The upregulation of nod and IAA genes was con rmed using reporter strains. Nod factor synthesis provides competitive advantage for colonization of rice and chemotaxis is required for colonization of S. cannabina but not of rice. ConclusionIRBG74 responded to S. cannabina and rice using speci c as well as common transcriptional changes. A mutant defective in Nod factor synthesis was outcompeted for rice colonization. Chemotaxis was required for colonization of S. cannabina but not for rice. IAA synthesis by IRBG74 could be a major mechanism of rice growth promotion. These results provide a foundation for further improvement of rhizobial interactions with rice and other cereals.