Rapid identification and typing of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and other <Emphasis Type="Italic">Yersinia</Emphasis> species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Background  

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.     

Functional Annotation,Genome Organization and Phylogeny of the Grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis>) Terpene Synthase Gene Family Based on Genome Assembly,FLcDNA Cloning,and Enzyme Assays

Background  

Terpenoids are among the most important constituents of grape flavour and wine bouquet, and serve as useful metabolite markers in viticulture and enology. Based on the initial 8-fold sequencing of a nearly homozygous Pinot noir inbred line, 89 putative terpenoid synthase genes (VvTPS) were predicted by in silico analysis of the grapevine (Vitis vinifera) genome assembly [1]. The finding of this very large VvTPS family, combined with the importance of terpenoid metabolism for the organoleptic properties of grapevine berries and finished wines, prompted a detailed examination of this gene family at the genomic level as well as an investigation into VvTPS biochemical functions.     

Surviving within the amoebal exocyst: the <Emphasis Type="Italic">Mycobacterium avium</Emphasis> complex paradigm

Background  

Most of environmental mycobacteria have been previously demonstrated to resist free-living amoeba with subsequent increased virulence and resistance to antibiotics and biocides. The Mycobacterium avium complex (MAC) comprises of environmental organisms that inhabit a wide variety of ecological niches and exhibit a significant degree of genetic variability. We herein studied the intra-ameobal location of all members of the MAC as model organisms for environmental mycobacteria.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Variable-number tandem-repeat markers for typing <Emphasis Type="Italic">Mycobacterium intracellulare</Emphasis> strains isolated in humans

Background  

Mycobacterium intracellulare, a species of the Mycobacterium avium complex, may be the cause of severe lung, lymphatic node, skin and bone/joint infections, as well as bacteriemia. The goal of this work was to identify Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) markers and to study their variability in a collection of isolates of M. intracellulare collected in humans. We studied 61 isolates collected in humans between 2001 and 2008, as well as the reference strain, M. intracellulare ATCC 13950.     

A Bacterial Ras-Like Small GTP-Binding Protein and Its Cognate GAP Establish a Dynamic Spatial Polarity Axis to Control Directed Motility

Regulated cell polarity is central to many cellular processes. We investigated the mechanisms that govern the rapid switching of cell polarity (reversals) during motility of the bacterium Myxococcus xanthus. Cellular reversals are mediated by pole-to-pole oscillations of motility proteins and the frequency of the oscillations is under the control of the Frz chemosensory system. However, the molecular mechanism that creates dynamic polarity remained to be characterized. In this work, we establish that polarization is regulated by the GTP cycle of a Ras-like GTPase, MglA. We initially sought an MglA regulator and purified a protein, MglB, which was found to activate GTP hydrolysis by MglA. Using live fluorescence microscopy, we show that MglA and MglB localize at opposite poles and oscillate oppositely when cells reverse. In absence of MglB, MglA-YFP accumulates at the lagging cell end, leading to a strikingly aberrant reversal cycle. Spatial control of MglA is achieved through the GAP activity of MglB because an MglA mutant that cannot hydrolyze GTP accumulates at the lagging cell end, despite the presence of MglB. Genetic and cell biological studies show that the MglA-GTP cycle controls dynamic polarity and the reversal switch. The study supports a model wherein a chemosensory signal transduction system (Frz) activates reversals by relieving a spatial inhibition at the back pole of the cells: reversals are allowed by Frz-activated switching of MglB to the opposite pole, allowing MglA-GTP to accumulate at the back of the cells and create the polarity switch. In summary, our results provide insight into how bacteria regulate their polarity dynamically, revealing unsuspected conserved regulations with eukaryots.