Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis.

Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an H2O2-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.

Interplay of Nitric Oxide Synthase (NOS) and SrrAB in Modulation of Staphylococcus aureus Metabolism and Virulence.

Staphylococcus aureus nitric oxide synthase (saNOS) is a major contributor to virulence, stress resistance, and physiology, yet the specific mechanism(s) by which saNOS intersects with other known regulatory circuits is largely unknown. The SrrAB two-component system, which modulates gene expression in response to the reduced state of respiratory menaquinones, is a positive regulator of nos expression. Several SrrAB-regulated genes were also previously shown to be induced in an aerobically respiring nos mutant, suggesting a potential interplay between saNOS and SrrAB. Therefore, a combination of genetic, molecular, and physiological approaches was employed to characterize a nos srrAB mutant, which had significant reductions in the maximum specific growth rate and oxygen consumption when cultured under conditions promoting aerobic respiration. The nos srrAB mutant secreted elevated lactate levels, correlating with the increased transcription of lactate dehydrogenases. Expression of nitrate and nitrite reductase genes was also significantly enhanced in the nos srrAB double mutant, and its aerobic growth defect could be partially rescued with supplementation with nitrate, nitrite, or ammonia. Furthermore, elevated ornithine and citrulline levels and highly upregulated expression of arginine deiminase genes were observed in the double mutant. These data suggest that a dual deficiency in saNOS and SrrAB limits S. aureus to fermentative metabolism, with a reliance on nitrate assimilation and the urea cycle to help fuel energy production. The nos, srrAB, and nos srrAB mutants showed comparable defects in endothelial intracellular survival, whereas the srrAB and nos srrAB mutants were highly attenuated during murine sepsis, suggesting that SrrAB-mediated metabolic versatility is dominant in vivo.

The t6A modification acts as a positive determinant for the anticodon nuclease PrrC, and is distinctively nonessential in Streptococcus mutans.

Endoribonuclease toxins (ribotoxins) are produced by bacteria and fungi to respond to stress, eliminate non-self competitor species, or interdict virus infection. PrrC is a bacterial ribotoxin that targets and cleaves tRNALysUUU in the anticodon loop. In vitro studies suggested that the post-transcriptional modification threonylcarbamoyl adenosine (t6A) is required for PrrC activity but this prediction had never been validated in vivo. Here, by using t6A-deficient yeast derivatives, it is shown that t6A is a positive determinant for PrrC proteins from various bacterial species. Streptococcus mutans is one of the few bacteria where the t6A synthesis gene tsaE (brpB) is dispensable and its genome encodes a PrrC toxin. We had previously shown using an HPLC-based assay that the S. mutans tsaE mutant was devoid of t6A. However, we describe here a novel and a more sensitive hybridization-based t6A detection method (compared to HPLC) that showed t6A was still present in the S. mutans ΔtsaE, albeit at greatly reduced levels (93% reduced compared with WT). Moreover, mutants in 2 other S. mutans t6A synthesis genes (tsaB and tsaC) were shown to be totally devoid of the modification thus confirming its dispensability in this organism. Furthermore, analysis of t6A modification ratios and of t6A synthesis genes mRNA levels in S. mutans suggest they may be regulated by growth phase.

Investigation of simulated microgravity effects on Streptococcus mutans physiology and global gene expression.

Astronauts have been previously shown to exhibit decreased salivary lysozyme and increased dental calculus and gingival inflammation in response to space flight, host factors that could contribute to oral diseases such as caries and periodontitis. However, the specific physiological response of caries-causing bacteria such as Streptococcus mutans to space flight and/or ground-based simulated microgravity has not been extensively investigated. In this study, high aspect ratio vessel S. mutans simulated microgravity and normal gravity cultures were assessed for changes in metabolite and transcriptome profiles, H2O2 resistance, and competence in sucrose-containing biofilm media. Stationary phase S. mutans simulated microgravity cultures displayed increased killing by H2O2 compared to normal gravity control cultures, but competence was not affected. RNA-seq analysis revealed that expression of 153 genes was up-regulated ≥2-fold and 94 genes down-regulated ≥2-fold during simulated microgravity high aspect ratio vessel growth. These included a number of genes located on extrachromosomal elements, as well as genes involved in carbohydrate metabolism, translation, and stress responses. Collectively, these results suggest that growth under microgravity analog conditions promotes changes in S. mutans gene expression and physiology that may translate to an altered cariogenic potential of this organism during space flight missions.

Recurrent incisional hernia, enterocutaneous fistula and loss of the substance of the abdominal wall: plastic with organic prosthesis, skin graft and VAC therapy. Clinical case.

Surgical wounds dehiscence is a serious post-operatory complication, with an incidence between 0.4% and 3.5%. Mortality is more than 45%. Complex wounds treatment may require a multidisciplinary management. VAC Therapy could be an alternative treatment regarding complex wound. VAC therapy has been recently introduced on skin's graft tissue management reducing skin graft rejection. The use of biological prosthesis has been tested in a contaminated field, better than synthetic meshes, which often need to be removed. The Permacol is more resistant to degradation by proteases due to its cross-links. Surgery is still considered the best treatment for digestive fistula. A 58 years old obese woman come to our attention, she was operated for an abdominal hernia. She had a post-operatory entero-cutaneous fistula. She was submitted to bowel resection, the anastomosis has been tailored and the hernia of the abdominal wall has been repaired with biological mesh for managing such condition. She had a wound dehiscence with loss of substance and the exposure of the biological prosthesis, nearly 20 cm diameter. She was treated first with antibiotic therapy and simple medications. In addiction, antibiotic therapy was necessary late associated to 7 months with advanced medications allowed a small reduction's defect. Because of its, treatment went on for two more months using VAC therapy. Antibiotic's therapy was finally suspended. The VAC therapy allowed the reduction of the gap, between skin and subcutaneous tissue, and the defect's size preparing a suitable ground for the skin graft. The graft, managed with the vac therapy, was necessary to complete the healing process.

Multiorgan female pelvic prolapse: pelvic organ prolapse suspension (P.O.P.S.) stapled transanal rectale resection (S.T.A.R.R.): new surgical tecniques and results.

AIM: To demonstrate the surgical treatment validity and the post-operative complication decrease. MATERIAL OF STUDY: Seventythree women who underwent P.O.P.S. + S.T.A.R.R. treatment, follow-up one year. RESULTS: We observed an important reduction or a completely disappearance about pre-operative signs and symptoms. DISCUSSION: We are aware that the proposed technique, if taken into account by urogynecologists, will raise several arguments and will raise many doubts and perplexities. For this reason we wanted develop a follow-up sufficiently long and many case studies with data to support our claims. CONCLUSIONS: We believe that the procedure proposed by us, given the results, was excellent in patients with multiorgan pelvic prolapse, especially with the vagina walls elongated and that retain a good trophism.