Conserved Signal Transduction Mechanisms and Dark Recovery Kinetic Tuning in the Pseudomonadaceae Short Light, Oxygen, Voltage (LOV) Protein Family.

Light-Oxygen-Voltage (LOV) flavoproteins transduce a light signal into variable signaling outputs via a structural rearrangement in the sensory core domain, which is then relayed to fused effector domains via α-helical linker elements. Short LOV proteins from Pseudomonadaceae consist of a LOV sensory core and N- and C-terminal α-helices of variable length, providing a simple model system to study the molecular mechanism of allosteric activation. Here we report the crystal structures of two LOV proteins from Pseudomonas fluorescens - SBW25-LOV in the fully light-adapted state and Pf5-LOV in the dark-state. In a comparative analysis of the Pseudomonadaceae short LOVs, the structures demonstrate light-induced rotation of the core domains and splaying of the proximal A'α and Jα helices in the N and C-termini, highlighting evidence for a conserved signal transduction mechanism. Another distinguishing feature of the Pseudomonadaceae short LOV protein family is their highly variable dark recovery, ranging from seconds to days. Understanding this variability is crucial for tuning the signaling behavior of LOV-based optogenetic tools. At 37 °C, SBW25-LOV and Pf5-LOV exhibit adduct state lifetimes of 1470 min and 3.6 min, respectively. To investigate this remarkable difference in dark recovery rates, we targeted three residues lining the solvent channel entrance to the chromophore pocket where we introduced mutations by exchanging the non-conserved amino acids from SBW25-LOV into Pf5-LOV and vice versa. Dark recovery kinetics of the resulting mutants, as well as MD simulations and solvent cavity calculations on the crystal structures suggest a correlation between solvent accessibility and adduct lifetime.

Characterization of Francisella species isolated from the cooling water of an air conditioning system

Strains of Francisella spp. were isolated from cooling water from an air conditioning system in Guangzhou, China. These strains are Gram negative, coccobacilli, non-motile, oxidase negative, catalase negative, esterase and lipid esterase positive. In addition, these bacteria grow on cysteine-supplemented media at 20 °C to 40 °C with an optimal growth temperature of 30 °C. Analysis of 16S rRNA gene sequences revealed that these strains belong to the genus Francisella. Biochemical tests and phylogenetic and BLAST analyses of 16S rRNA, rpoB and sdhA genes indicated that one strain was very similar to Francisella philomiragia and that the other strains were identical or highly similar to the Francisella guangzhouensis sp. nov. strain 08HL01032 we previously described. Biochemical and molecular characteristics of these strains demonstrated that multiple Francisella species exist in air conditioning systems.

Porfiria variegata en Chile: identificación de mutaciones en el gen protoporfirinógeno oxidasa y su implicancia diagnóstica / Identification of mutations in the protoporphyrin oxidase gene and its diagnostic implications in porphyria variegata in Chile

Variegate porphyria (VP) results from a hereditary deficiency of protoporphyrinogen oxidase (PPOX) that is transmitted in an autosomal dominan fashion. The diagnosis is based on the clinical symptoms and is confirmed biochemically. Sometimes, however, these diagnostic tools reveal limitations in establishing the definitive diagnosis of the prevailing type of acute porphyria. In these patients, molecular genetic analyses can be useful. We performed molecular genetic studies in 13 Chilean families by PCR amplification of the PPOX gene, conformation sensitive gel electrophoresis, and automated DNA sequencing. In five symptomatic patients from different families, respectively, the biochemical data confirmed the diagnosis of VP. In seven other families, however, the biochemical studies were not conclusive. Furthermore, the original biochemical analysis in one clinically severely affected patient from a further family even suggested the diagnosis of erythropoietic protoporphyria (EPP). Beside the respective index patients, we studied 78 asymptomatic family members and 50 healthy, unrelated individuals for control purposes. In five families, the previous diagnosis of VP could be confirmed genetically. Further, half of the asymptomatic relatives revealed a mutation in the PPOX gene, consisting of three missense mutations and two deletion mutations. Mutation R168H that had been already described previously in German VP families was found in a Chilean family of German origin. Further, two novel missense mutations, designated L74P and G232S, could be detected. In four Chilean families, we found the deletion 1330deICT that had also been previously described in three Swedish VP families. The second deletion, 1239delTACAC, has not been described anywhere else but Chile and could be identified in seven families. One patient who was initially diagnosed with EPP turned out to be a compound heterozygote for mutations on both alíeles of the PPOX gene. In conclusion, our molecular genetic analyses unequivocally confirmed the diagnosis of VP in seven families who originally had revealed inconclusive biochemical data. Further, early genetic analysis allows for the identification of asymptomatic mutation carriers, thereby offering the possibility of adequate counselling and the prevention of potentially life-threatening acute porphyric attacks.

La porfiria variegata (PV), enfermedad de origen genético con forma de herencia autosómica dominante, se debe a deficiencia en la actividad protoporfirinógeno oxidasa (PPOX). Su diagnóstico se basa en antecedentes clínicos y se confirma con análisis bioquímicos. Éstos, en algunos casos, pueden presentar limitaciones para establecer el diagnóstico definitivo de la variedad de porfiria aguda, situación en que el estudio genético molecular puede resultar útil. Se efectuó estudio genético en trece familias chilenas usando amplificación del gen PPOX por PCR, electroforesis conformacional y secuenciación automática de DNA. Cinco de estas familias incluían pacientes índices sintomáticos con diagnóstico bioquímico establecido de PV; otras siete familias incluían pacientes índices con estudio bioquímico no concluyente de la variedad de porfiria aguda y, finalmente, una familia con diagnóstico previo de protoporfiria eritropoyética (PPE). Además, se estudiaron 78 familiares asintomáticos y 50 personas sanas, no relacionadas, como controles. En cinco familias el estudio genético confirmó el diagnóstico bioquímico previo de PV. El 50% de los familiares asintomáticos resultaron ser portadores de una mutación en el gen PPOX. Se identificaron tres mutaciones por sustitución de bases: la R168H, descrita en familias de origen alemán y dos nuevas mutaciones, designadas L74P y G232S. También se identificaron dos mutaciones por deleción de bases designadas 1330delCT y la 1239delTACAC. La primera, que había sido descrita previamente en tres familias suecas, se encontró en cuatro familias chilenas. La segunda se encontró en siete familias y no ha sido descrita previamente. El estudio genético permitió mostrar que un paciente que originalmente fue diagnosticado con PPE correspondía a un heterocigoto compuesto para dos mutaciones en el gen PPOX. En conclusión, los estudios moleculares permitieron confirmar el diagnóstico de PV en cinco familias, efectuar diagnóstico de PV en familias en las cuales los datos bioquímicos no eran concluyentes, corregir el diagnóstico original en una familia e identificar portadores asintomáticos entre los familiares de los pacientes índices. Los estudios genéticos moleculares ayudan a realizar un adecuado consejo genético a pacientes y familiares y hace posible practicar prevención de las crisis agudas de porfiria, las que son potencialmente mortales.