The RepA and RepB autorepressors and TraR play opposing roles in the regulation of a Ti plasmid repABC operon

The replicator regions of the Ti plasmids of Agrobacterium tumefaciens belong to the repABC family of replication and partitioning systems, members of which are widely distributed among alpha proteobacteria. In the region upstream of the octopine-type Ti plasmid repABC operon, three promoters were recently shown to be activated by the LuxR-type regulator TraR. Activation of these promoters by TraR led to enhanced rep gene expression and increased Ti plasmid copy number. Here we describe a fourth promoter, designated P4. This promoter lies directly upstream of repA and is not regulated by TraR. The promoter was localized by subcloning and demonstrated to be strongly autorepressed. RepA is the major cis-acting autorepressor of this promoter, though RepB enhanced repression and was essential for RepA-mediated repression in trans. Purified RepA bound to an approximately 70-nucleotide operator site overlapping the P4 promoter and extending well downstream. Binding affinity was increased by adenosine di- and tri-phosphates and also by purified RepB. Activation of P1, P2, and P3 enhanced the activity of P4, suggesting that P4 somehow communicates with the upstream promoters. These findings demonstrate that both autoinduction and autorepression play critical and opposing roles in regulating repABC expression and hence in the replication, stability and copy number of the Ti plasmid.     

Allometric growth of the proglottids and strobila of the tapeworm, Hymenolepis diminuta

HYMENOLEPIS DIMINUTA: adults were killed and fixed in fully extended positions, and the total lengths of the strobilae, numbers of proglottids, and distances of proglottids along the strobilae were determined. The relationship of proglottid number to distance along the strobila was exponential. Beginning at proglottid 100 (P100), the lengths and widths of proglottids at 100 proglottid intervals were determined, and the surfaces areas were calculated. The relationships of proglottid length and width to proglottid number were linear, but the relationship of proglottid number to surface area (SA) was exponential. The volumes of proglottids were calculated, and the relationship of volume (V) to proglottid number was exponential. The relationship of surface area to volume ratio (SAVR) to proglottid number was exponential; at the anterior end of the worm (P100), the SAVR was 14.6, while at the posterior end of the worm (P1300) the ratio was 4.2. A single exponential equation describing the relationships among proglottid number, SA, and V was derived.     

Effect of colony morphology variation of <Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis> on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro

Background  

Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays.     

<Emphasis Type="Italic">Burkholderia</Emphasis> Hep_Hag autotransporter (BuHA) proteins elicit a strong antibody response during experimental glanders but not human melioidosis

Background  

The bacterial biothreat agents Burkholderia mallei and Burkholderia pseudomallei are the cause of glanders and melioidosis, respectively. Genomic and epidemiological studies have shown that B. mallei is a recently emerged, host restricted clone of B. pseudomallei.     

A Replicative Plasmid Vector Allows Efficient Complementation of Pathogenic Leptospira Strains

Leptospirosis, an emerging zoonotic disease, remains poorly understood because of a lack of genetic manipulation tools available for pathogenic leptospires. Current genetic manipulation techniques include insertion of DNA by random transposon mutagenesis and homologous recombination via suicide vectors. This study describes the construction of a shuttle vector, pMaORI, that replicates within saprophytic, intermediate, and pathogenic leptospires. The shuttle vector was constructed by the insertion of a 2.9-kb DNA segment including the parA, parB, and rep genes into pMAT, a plasmid that cannot replicate in Leptospira spp. and contains a backbone consisting of an aadA cassette, ori R6K, and oriT RK2/RP4. The inserted DNA segment was isolated from a 52-kb region within Leptospira mayottensis strain 200901116 that is not found in the closely related strain L. mayottensis 200901122. Because of the size of this region and the presence of bacteriophage-like proteins, it is possible that this region is a result of a phage-related genomic island. The stability of the pMaORI plasmid within pathogenic strains was tested by passaging cultures 10 times without selection and confirming the presence of pMaORI. Concordantly, we report the use of trans complementation in the pathogen Leptospira interrogans. Transformation of a pMaORI vector carrying a functional copy of the perR gene in a null mutant background restores the expression of PerR and susceptibility to hydrogen peroxide comparable to that of wild-type cells. In conclusion, we demonstrate the replication of a stable plasmid vector in a large panel of Leptospira strains, including pathogens. The shuttle vector described will expand our ability to perform genetic manipulation of Leptospira spp.     

Bronchoabsorption; a novel bronchoscopic technique to improve biomarker sampling of the airway

Background

Current techniques used to obtain lung samples have significant limitations and do not provide reproducible biomarkers of inflammation. We have developed a novel technique that allows multiple sampling methods from the same area (or multiple areas) of the lung under direct bronchoscopic vision. It allows collection of mucosal lining fluid and bronchial brushing from the same site; biopsy samples may also be taken. The novel technique takes the same time as standard procedures and can be conducted safely.

Methods

Eight healthy smokers aged 40–65 years were included in this study. An absorptive filter paper was applied to the bronchial mucosa under direct vision using standard bronchoscopic techniques. Further samples were obtained from the same site using bronchial brushings. Bronchoalveolar lavage (BAL) was obtained using standard techniques. Chemokine (C-C Motif) Ligand 20 (CCL20), CCL4, CCL5, Chemokine (C-X-C Motif) Ligand 1 (CXCL1), CXCL8, CXCL9, CXCL10, CXCL11, Interleukin 1 beta (IL-1β), IL-6, Vascular endothelial growth factor (VEGF), Matrix metalloproteinase 8 (MMP-8) and MMP-9 were measured in exudate and BAL. mRNA was collected from the bronchial brushings for gene expression analysis.

Results

A greater than 10 fold concentration of all the biomarkers was detected in lung exudate in comparison to BAL. High yield of good quality RNA with RNA integrity numbers (RIN) between 7.6 and 9.3 were extracted from the bronchial brushings. The subset of genes measured were reproducible across the samples and corresponded to the inflammatory markers measured in exudate and BAL.

Conclusions

The bronchoabsorption technique as described offers the ability to sample lung fluid direct from the site of interest without the dilution effects caused by BAL. Using this method we were able to successfully measure the concentrations of biomarkers present in the lungs as well as collect high yield mRNA samples for gene expression analysis from the same site. This technique demonstrates superior sensitivity to standard BAL for the measurement of biomarkers of inflammation. It could replace BAL as the method of choice for these measurements. This method provides a systems biology approach to studying the inflammatory markers of respiratory disease progression.

Trial registration

NHS Health Research Authority (13/LO/0256).     

Identification of genes encoding hypothetical proteins in open-reading frame expressed sequence tags from mammalian stages of Trypanosoma cruzi

Approximately 50% of the predicted protein-coding genes of the Trypanosoma cruzi CL Brener strain are annotated as hypothetical or conserved hypothetical proteins. To further characterize these genes, we generated 1161 open-reading frame expressed sequence tags (ORESTES) from the mammalian stages of the VL10 human strain. Sequence clustering resulted in 435 clusters, consisting of 339 singletons and 96 contigs. Significant matches to the T. cruzi predicted gene database were found for ~94% contigs and ~69% singletons. These included genes encoding surface proteins, known to be intensely expressed in the parasite mammalian stages and implicated in host cell invasion and/or immune evasion mechanisms. Among 151 contigs and singletons with similarity to predicted hypothetical protein-coding genes and conserved hypothetical protein-coding genes, 83% showed no match with T. cruzi EST and/or proteome databases. These ORESTES are the first experimental evidence that the corresponding genes are in fact transcribed. Sequences with no significant match were searched against several T. cruzi and National Center for Biotechnology Information non-redundant sequence databases. The ORESTES analysis indicated that 124 predicted conserved hypothetical protein-coding genes and 27 predicted hypothetical protein-coding genes annotated in the CL Brener genome are transcribed in the VL10 mammalian stages. Six ORESTES annotated as hypothetical protein-coding genes showing no match to EST and/or proteome databases were confirmed by Northern blot in VL10. The generation of this set of ORESTES complements the T. cruzi genome annotation and suggests new stage-regulated genes encoding hypothetical proteins.