Interestingly, many genes related to bacterial motility, such as flagellar biosynthesis and chemotaxis genes, also were identified as being down-regulated in vivo in our screen. Concentrations of medium additives when necessary were as follows: 100 μg/μl rifampicin; 100 μg/μl ampicillin; 30 μg/mL gentamicin, 1 mM isopropyl b-D-1-thiogalactopyranoside (IPTG); and 0.05% arabinose. LtetO-1 The V. cholerae rugose phenotype represents a fully virulent survival form of the organism [44] that can persist in the natural environment by using its ability to form biofilms [31,32]. P The environmental conditions which induce the state probably differ from bacterium to bacterium and it is likely that the physiology involved varies among different cells.

M.

5

Writing – original draft, Affiliations

The experiment in S1D Fig was performed using pre-cast SuperSep Phos-Tag gels (Wako).

16S ribosomal RNA was used for an internal control in all reactions. A study finds that imposing a tax on orbiting satellites could increase the value of the satellite industry from $600 billion to $3 trillion by 2040 by decreasing collision risks and space debris. We developed a FACS-based screening method to identify V. cholerae genes that are expressed in vitro and repressed during infection of the infant mouse, a model mammalian host. PLoS Pathog 16(8): (ii) In the native phosphoregulatory system, autokinase activity leads to VpsO phosphorylation, higher order oligomer dissociation, and a reduction in VPS production. Colwell In the crystal, domain swapping in the dimer occurs through exchange of the C-terminal α-helix (α5).

Qualitatively, the rugose, vpsOK551A, and ΔvpsU strains displayed similar spatiotemporal patterns for both the nearest-neighbor distance and the local nematic order. The confocal microscope used in this study was funded by NIH IS10 OD023528 (FHY). We also purified a domain in which the predicted active site lysine residue (K551) was replaced with an alanine (VpsO-503K551A). (, Steinert The ΔvpsOΔctxAB pBAD B vpsO-Myc/His strain was used to purify full-length VpsO-Myc/His from V. cholerae to determine its native phosphorylation sites. A. (B) Representative phase contrast images of stationary phase rugose and ΔvpsO cells taken at 63x magnification. However, FACS analysis of intestines from uninfected mice did not reveal a GFP+ population (data not shown). R.M. Bacteria were grown in LB at 37°C or incubated in 5-day-old infant mice for 12 h and were analyzed by flow cytometry. We detected expression of all VPS cluster proteins except VpsI, and of these proteins only VpsO was observed to be tyrosine phosphorylated under the conditions tested (S5 Fig and S3 Table). Reasoner We found that upon incubation with ATP the wild-type version of the VpsO-503 domain underwent a mobility shift consistent with autophosphorylation, while both the VpsO-503Y717A, Y720A, Y721A, Y723A, Y726A, Y727A and the VpsO-503K551A mutated domains showed no mobility shift and therefore no autophosphorylation in this assay (Fig 2A). Chapman (A) Colony corrugation phenotypes of complementation strains after 5 days of growth at 25°C. This observation suggests that the production of rounded cells is induced only when cells are continuously supplied with fresh nutrients under flow conditions.

The results are representative of three experiments ± SD. The two non-VPS producing strains, ΔvpsO and vpsOE519A, R522A, R525A, had the highest nearest-neighbor distance, and they only formed a monolayer of cells on the glass surface, without 3D structure.

Peptides were eluted with 40% acetonitrile in 0.5% acetic acid followed by 80% acetonitrile in 0.5% acetic acid. R.W. *****, p < 0.0005 by one-way Anova multiple comparisons test. Korber Since then, studies have revealed the existence of V. cholerae as free-living bacteria or in association with phytoplankton, zooplankton, crustaceans and mollusks in coastal and estuarine environments [4–7]. Collectively, these findings support our conclusion that autophosphorylation of the VpsO C-terminal tail increases VpsO stability, and that the position of tyrosine phosphorylation as well as number of phosphorylated tyrosine residues are critical for VPS production. Jr. [51], Wai et al. V. cholerae strains were grown in LB or AKI medium and assayed for MSHA production by hemagglutination and TcpA production by a Western blot.

Investigation, Emody R.A. Approximately 105 mid-logarithmic phase bacteria were collected by centrifugation and resuspended in 100 μl of Krebbs-Ringer-Tris (KRT) buffer (42). B. West M.R. To identify V. cholerae-repressed genes in vivo, we constructed a V. cholerae strain containing a P After washing, chambers were stained for live/dead cells with SYTO 9 (live cells) and propidium iodide (dead cells) and suspended in DASW for 15 minutes at room temperature (BacLight Bacterial Viability Kit, Invitrogen).

F.L. 2 Motita -gfp was cloned into the plasmid pCVD442 (37) and integrated into the lacZ locus of the C6706 chromosome, resulting in the strain JZV338. We next analyzed VPS levels in these strains. (, Ostling Sack

Unfortunately, we still know little about the role of V. cholerae in forming biofilms, how biofilms may improve survival of this organism in the environment and the epidemiology of the disease in human populations. (A) Diagram of the VpsO protein highlighting the positions of the ExxRxxR motifs. Writing – original draft, Roles In contrast, the VpsO-503E519A, R522A, R525A protein used for crystallization lacks the N-terminal extension found in other BY-kinases, and alternatively, its α1 helix is longer at its N-terminus. We found that tyrosine phosphorylation enhances stability of VpsO. Although many studies have focused on understanding the starvation response of marine vibrios, there is still limited understanding of how V. cholerae responds to starvation stress. Interestingly, the respiratory bacterium Bordetella bronchiseptica employs the BvgAS two-component system to enhance virulence by both activating and repressing genes during colonization (30).

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