Microbial flavohaemoglobins are proteins with homology to haemoglobins from higher organisms, but clearly linked to nitric oxide (NO) metabolism by bacteria and yeast. hmp mutant strains of several bacteria are hypersensitive to NO and related compounds and hmp genes are up-regulated by the presence of NO. The regulatory mechanisms involved in hmp induction by NO and the superoxide-generating agent, methyl viologen (paraquat; PQ), are complex, but progressively being resolved. Here we show for the first time that, in Salmonella enterica serovar Typhimurium, hmp transcription is increased on exposure to PQ and demonstrate that RamA, a homologue of MarA is responsible for most of the hmp paraquat regulation. In addition we demonstrate NO-dependent elevation of Salmonella hmp transcription and Hmp accumulation. In both Escherichia coli and Salmonella modest transcriptional repression of hmp is exerted by the iron responsive transcriptional repressor Fur. Finally, in contrast to previous reports, we show that in E. coli and Salmonella, hmp induction by both paraquat and sodium nitroprusside is further elevated in a fur mutant background, indicating that additional regulators are implicated in this control process.

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 M NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.

e19518 Background: Microbiome is a challenging study area due to its influence on the multiple host functions. The majority of human-associated microbes reside within the colon. Our understanding about the complex interplay between host and environmental factors to shaping the microbiota is evolving rapidly AL is an unfavorable prognosis disease whose only cure possibility is the allo-HSCT. This procedure uses high doses of chemotherapy and multiple drugs such as antibiotics, antiviral, antifungals, and immunosuppressants that damage the mucous membranes and alter the intestinal microbiome balance. These events have been linked to bacterial resistance, relapse risk, Graft Versus Host Disease (GVHD), and poor Overall Survival (OS). The present study’s objective was to identify the intestinal microbiome bacteria groups during allo-HSCT and to evaluate their impact on patients outcome. Methods: Observational and prospective study was performed. Eleven patients with acute leukemia under alloHSCT and 11 health control (relatives) were enrolled. Gut faecal samples were collected in both groups; three for patients (at income day (ID), neutropenic period (N) and 30 days after discharge (+30D) and one in healthy donor (HD) at income day of their relatives. Bacterial 16S rRNA gene sequences were characterized by illumina and QUIIME 2. Biodiversity of microbiome was evaluated by OTUS, Shannon index and dominance. This proyect was supported by CONACyT. Results: We analyze 11 patients, 55% were male, with a median of 25 years-old at allo-HSCT. 7/10 received a HLA-identical and 4/10 an haploidentical HSCT. 82% had GVHD (I-IV), 1/11 died (two due to infections-GVHD and one of disease relapse). We analyzed 44 samples. There are no difference between healthy control group and income day patients’ samples. Statistical differences in the patients’ microbiome were identified among HSCT moments. According OTUS and Shannon Index the biodiversity decrease at neutropenia, and increase at day +30 outcome but it doesn’t represent a complete recovery. Greater bacterial dominance was observed in neutropenia period. (Table). 3/10 patients who died didn’t recovery biodiversity at day +30. Conclusions: Our results suggest that poor microbiome biodiversity recovery could be a worst prognostic [Table: see text]