Reciprocal control of motility and biofilm formation by the PdhS2 two-component sensor kinase of Agrobacterium tumefaciens.

A core regulatory pathway that directs developmental transitions and cellular asymmetries in Agrobacterium tumefaciens involves two overlapping, integrated phosphorelays. One of these phosphorelays putatively includes four histidine sensor kinase homologues, DivJ, PleC, PdhS1 and PdhS2, and two response regulators, DivK and PleD. In several different alphaproteobacteria, this pathway influences a conserved downstream phosphorelay that ultimately controls the phosphorylation state of the CtrA master response regulator. The PdhS2 sensor kinase reciprocally regulates biofilm formation and swimming motility. In the current study, the mechanisms by which the A. tumefaciens sensor kinase PdhS2 directs this regulation are delineated. PdhS2 lacking a key residue implicated in phosphatase activity is markedly deficient in proper control of attachment and motility phenotypes, whereas a kinase-deficient PdhS2 mutant is only modestly affected. A genetic interaction between DivK and PdhS2 is revealed, unmasking one of several connections between PdhS2-dependent phenotypes and transcriptional control by CtrA. Epistasis experiments suggest that PdhS2 may function independently of the CckA sensor kinase, the cognate sensor kinase for CtrA, which is inhibited by DivK. Global expression analysis of the pdhS2 mutant reveals a restricted regulon, most likely functioning through CtrA to separately control motility and regulate the levels of the intracellular signal cyclic diguanylate monophosphate (cdGMP), thereby affecting the production of adhesive polysaccharides and attachment. We hypothesize that in A. tumefaciens the CtrA regulatory circuit has expanded to include additional inputs through the addition of PdhS-type sensor kinases, likely fine-tuning the response of this organism to the soil microenvironment.

A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens.

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.

Beryllium detection in human lung tissue using electron probe X-ray microanalysis.

Chronic berylliosis is an uncommon disease that is caused by the inhalation of beryllium particles, dust, or fumes. The distinction between chronic berylliosis and sarcoidosis can be difficult both clinically and histologically, as both entities can have similar presentations and exhibit nonnecrotizing granulomatous inflammation of the lungs. The diagnosis of chronic berylliosis relies on a history of exposure to beryllium, roentgenographic evidence of diffuse nodular disease, and demonstration of beryllium hypersensitivity by ancillary studies, such as lymphocyte proliferation testing. Additional support may be gained by the demonstration of beryllium in lung tissue. Unlike other exogenous particulates, such as asbestos, detection of beryllium in human lung tissue is problematic. The low atomic number of beryllium usually makes it unsuitable for conventional microprobe analysis. We describe a case of chronic berylliosis in which beryllium was detected in lung tissue using atmospheric thin-window energy-dispersive X-ray analysis (ATW EDXA). A woman with a history of occupational exposure to beryllium at a nuclear weapons testing facility presented with progressive cough and dyspnea and a nodular pattern on chest roentgenograph. Open lung biopsy showed nonnecrotizing granulomatous inflammation that was histologically indistinguishable from sarcoidosis. Scanning electron microscopy and ATW EDXA demonstrated particulates containing beryllium within the granulomas. This application of EDXA offers significant advantages over existing methods of beryllium detection in that it is nondestructive, more widely available, and can be performed using routine paraffin sections.