Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci.

BACKGOUND: Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy. METHODS: We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2. RESULTS: All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules. CONCLUSIONS: Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications. GENERAL SIGNIFICANCE: Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

Mechanisms, Detection, and Relevance of Protein Acetylation in Prokaryotes.

Posttranslational modification of a protein, either alone or in combination with other modifications, can control properties of that protein, such as enzymatic activity, localization, stability, or interactions with other molecules. N-ε-Lysine acetylation is one such modification that has gained attention in recent years, with a prevalence and significance that rival those of phosphorylation. This review will discuss the current state of the field in bacteria and some of the work in archaea, focusing on both mechanisms of N-ε-lysine acetylation and methods to identify, quantify, and characterize specific acetyllysines. Bacterial N-ε-lysine acetylation depends on both enzymatic and nonenzymatic mechanisms of acetylation, and recent work has shed light into the regulation of both mechanisms. Technological advances in mass spectrometry have allowed researchers to gain insight with greater biological context by both (i) analyzing samples either with stable isotope labeling workflows or using label-free protocols and (ii) determining the true extent of acetylation on a protein population through stoichiometry measurements. Identification of acetylated lysines through these methods has led to studies that probe the biological significance of acetylation. General and diverse approaches used to determine the effect of acetylation on a specific lysine will be covered.

Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map-based cloning of AvrLm6.

Map-based cloning of avirulence genes of the AvrLml-2-6 cluster was recently undertaken in Leptosphaeria maculans and led to the identification of AvrLm1. The ensuing chromosome walk toward AvrLm6 resulted in the delineation of a 562-kb bacterial artificial chromosome (BAC) clone contig in an avirulent isolate. Following sequencing of the contig and sequence comparison with a virulent isolate, four AvrLm6 candidate genes were identified. Complementation of the virulent isolate with the four candidates was performed and one gene was found to fully restore the avirulent phenotype on Rlm6 oilseed rape genotypes. AvrLm6 was found to be located in the same genome context as AvrLml, because it is a solo gene surrounded by 85 and 48 kb of degenerated repeats on its 5' and 3' sides, respectively. AvrLm6 is an orphan gene encoding a small, potentially secreted, cysteine-rich protein. Comparison of AvrLm1 and AvrLm6 expressions by quantitative reverse-transcription polymerase chain reaction revealed that both genes are highly overexpressed during primary leaf infection. Using RNA interference, decreasing expression of AvrLm6 was shown to result in virulence toward Rlm6 genotypes whenever the expression was reduced by more than 60% compared with the wild-type isolate.

New Avirulence Genes in the Phytopathogenic Fungus Leptosphaeria maculans.

ABSTRACT Leptosphaeria maculans, the causal agent of stem canker of oilseed rape (Brassica napus), develops gene-for-gene interactions with oilseed rape, and four L. maculans avirulence (AVR) genes (AvrLm1, AvrLm2, AvrLm4, and alm1) were previously genetically characterized. Based on the analysis of progeny of numerous in vitro crosses between L. maculans isolates showing either already characterized or new differential interactions, this work aims to provide an overview of the AVR genes that may specify incompatibility toward B. napus and the related species B. juncea and B. rapa. Two novel differential interactions were thus identified between L. maculans and B. napus genotypes, one of them corresponding to a complete resistance to European races of L. maculans. In both cases, a single gene control of avirulence was established (genes AvrLm3 and AvrLm7). Similarly, a single gene control of avirulence toward a B. rapa genotype, also resistant to European L. maculans isolates, was demonstrated (gene AvrLm8). Finally, a digenic control of avirulence toward B. juncea was established (genes AvrLm5 and AvrLm6). Linkage analyses demonstrated that at least four unlinked L. maculans genomic regions, including at least one AVR gene cluster (AvrLm1-AvrLm2-AvrLm6), are involved in host specificity. The AvrLm3-AvrLm4-AvrLm7 region may correspond either to a second AVR gene cluster or to a multiallelic AVR gene.

[Chondrosarcoma of the orbit. Report of a case]. / Condrossarcoma de ossos da orbita. Relato de um caso

A case of chondrosarcoma of the orbit is reported. The patient complained of marked deterioration of her vision, pain and presented exophatalmus of the left eye. Chondrosarcoma of the orbit are rare and this is the first case reported in the Brazilian literature. Diagnostic and prognostic are discussed.

[Blastomycotic granuloma of the cervical spine. Report of a case]. / Granuloma blastomicotico na medula cervical. Registro de um caso

A case of blastomycotic granuloma of the spinal cord is reported. The patient had a Brown-Séquard syndrome. He was submitted to myelography and he was underwent a cervical laminectomy. The mass was removed being lately confirmed by histology as a blastomycotic granuloma. Despite the medical treatment with Amphotericin B and physical therapy there was no improvement of the condition.