In this study, a DeoR/GlpR-type transcription factor was investigated for its potential role as a global regulator of sugar metabolism in haloarchaea, using Haloferax volcanii as a model organism. Common to a number of haloarchaea and Gram-positive bacterial species, the encoding glpR gene was chromosomally linked with genes of sugar metabolism. In H. volcanii, glpR was cotranscribed with the downstream phosphofructokinase (PFK; pfkB) gene, and the transcript levels of this glpR-pfkB operon were 10-to 20-fold higher when cells were grown on fructose or glucose than when they were grown on glycerol alone. GlpR was required for repression on glycerol based on significant increases in the levels of PFK (pfkB) transcript and enzyme activity detected upon deletion of glpR from the genome. Deletion of glpR also resulted in significant increases in both the activity and the transcript (kdgK1) levels of 2-keto-3-deoxy-D-gluconate kinase (KDGK), a key enzyme of haloarchaeal glucose metabolism, when cells were grown on glycerol, compared to the levels obtained for media with glucose. Promoter fusions to a ␤-galactosidase bgaH reporter revealed that transcription of glpR-pfkB and kdgK1 was modulated by carbon source and GlpR, consistent with quantitative reverse transcription-PCR (qRT-PCR) and enzyme activity assays. The results presented here provide genetic and biochemical evidence that GlpR controls both fructose and glucose metabolic enzymes through transcriptional repression of the glpR-pfkB operon and kdgK1 during growth on glycerol.The archaeal basal transcriptional machinery closely resembles the eucaryal RNA polymerase II (RNAP II) apparatus. Along with a multisubunit RNAP (46), archaea encode two basal transcription factors, TATA-binding protein (TBP) and transcription factor B (TFB), which are homologs of the eucaryal TBP and general transcription factor TFIIB, respectively (5, 39). Although archaeal transcriptional components are fundamentally eukaryote-like in nature (35), the majority of candidate transcriptional regulators are homologous to bacterial activators and repressors (2, 26). Only a few archaeal candidate regulators resemble eukaryotic gene-specific transcription factors, one of the best characterized of which is GvpE, an activator of gas vesicle biosynthesis in haloarchaea which resembles the eukaryotic basic leucine zipper proteins (25, 31). While bioinformatics predicts many candidate archaeal regulators, only a limited number have been characterized at the molecular level, most of which are from hyperthermophiles (4,13,23,24,27,42). Molecular data pertaining to haloarchaeal transcriptional regulation, specifically regulators of carbon utilization, are severely limited. Only a few global regulators, namely, transcription factors (8, 11, 36), have been implicated in regulating carbon utilization in haloarchaea. Specifically, in Halobacterium salinarium, pairs of general transcription factors TBP and TFB control gene clusters (8, 11), and transcription factor TrmB regulates diverse metabolic pathwa...

The distribution of vesicular stomatitis virus (VSV) nucleocapsids in the cytoplasm of infected cells was analyzed by scanning confocal fluorescence microscopy using a newly developed quantitative approach called the border-to-border distribution method. Nucleocapsids were located near the cell nucleus at early times postinfection (2 h) but were redistributed during infection toward the edges of the cell. This redistribution was inhibited by treatment with nocodazole, colcemid, or cytochalasin D, indicating it is dependent on both microtubules and actin filaments. The role of actin filaments in nucleocapsid mobility was also confirmed by live-cell imaging of fluorescent nucleocapsids of a virus containing P protein fused to enhanced green fluorescent protein. However, in contrast to the overall redistribution in the cytoplasm, the incorporation of nucleocapsids into virions as determined in pulse-chase experiments was dependent on the activity of actin filaments with little if any effect on inhibition of microtubule function. These results indicate that the mechanisms by which nucleocapsids are transported to the farthest reaches of the cell differ from those required for incorporation into virions. This is likely due to the ability of nucleocapsids to follow shorter paths to the plasma membrane mediated by actin filaments. IMPORTANCENucleocapsids of nonsegmented negative-strand viruses like VSV are assembled in the cytoplasm during genome RNA replication and must migrate to the plasma membrane for assembly into virions. Nucleocapsids are too large to diffuse in the cytoplasm in the time required for virus assembly and must be transported by cytoskeletal elements. Previous results suggested that microtubules were responsible for migration of VSV nucleocapsids to the plasma membrane for virus assembly. Data presented here show that both microtubules and actin filaments are responsible for mobility of nucleocapsids in the cytoplasm, but that actin filaments play a larger role than microtubules in incorporation of nucleocapsids into virions. N ucleocapsids of negative-strand RNA viruses must be transported from their sites of assembly in the cytoplasm to sites of virus budding from host membranes (1). For example, the nucleocapsids of vesicular stomatitis virus (VSV) behave as random coils with a hydrodynamic radius of approximately 90 nm (2), which is too large to diffuse through the cytoplasm in the time required for virus assembly (3). Transport of nucleocapsids to the membrane after assembly in the cytoplasm has been proposed to occur primarily along microtubules (4). The goal of the experiments presented here was to further test mechanisms of nucleocapsid transport by evaluating both microtubule-dependent and actin-dependent transport using recently developed analytical tools.Actin filaments and microtubules have a general orientation in which the growing (plus) end is oriented toward the cell periphery and the minus end is oriented toward the center of the cell (5). Assembly of microtubules is usually...

Comparing the distribution of cytoplasmic particles and organelles between different experimental conditions can be challenging due to the heterogeneous nature of cell morphologies. The border-to-border distribution method was created to enable the quantitative analysis of fluorescently labeled cytoplasmic particles and organelles of multiple cells from images obtained by confocal microscopy. The method consists in four steps: (1) imaging of fluorescently labeled cells, (2) division of the image of the cytoplasm into radial segments, (3) selection of segments of interest and (4) population analysis of fluorescence intensities at the pixel level either as a function of distance along the selected radial segments or as a function of angle around an annulus. The method was validated using the well-characterized effect of brefeldin A (BFA) on the distribution of the vesicular stomatitis virus G protein, in which intensely labeled Golgi membranes are redistributed within the cytoplasm. Surprisingly, in untreated cells, the distribution of fluorescence in Golgi membrane-containing radial segments was similar to the distribution of fluorescence in other G protein-containing segments, indicating that the presence of Golgi membranes did not shift the distribution of G protein towards the nucleus compared to the distribution of G protein in other regions of the cell. Treatment with BFA caused only a slight shift in the distribution of the brightest G protein-containing segments, which had a distribution similar to that in untreated cells. Instead, the major effect of BFA was to alter the annular distribution of G protein in the perinuclear region.