Cell Cycle Control of a Holdfast Attachment Gene in Caulobacter crescentus

Self Cite

SummaryThe differentiating bacterium Caulobacter crescentus produces two different cell types at each cell division, a motile swarmer cell and an adhesive stalked cell. The stalked cell harbours a stalk, a thin cylindrical extension of the cell surface. The tip of the stalk is decorated with a holdfast, an adhesive organelle composed at least in part of polysaccharides. The synthesis of the stalk and holdfast occur at the same pole during swarmer cell differentiation. Mutations in the hfaABDC gene cluster had been shown to disrupt the attachment of the holdfast to the tip of the stalk, but the role of individual genes was unknown. We used lacZ fusions of various DNA fragments from the hfaABDC region to show that these genes form an operon. In order to analyse the relative contribution of the different genes to holdfast attachment, mutations were constructed for each gene. hfaC was not required for holdfast attachment or binding to surfaces. The hfaA and hfaD mutants shed some holdfast material into the surrounding medium and were partially deficient in binding to surfaces. Unlike hfaA and hfaB mutants, hfaD mutants were still able to form rosettes efficiently. Cells with insertions in hfaB were unable to bind to surfaces, and lectin binding studies indicated that the hfaB mutants had the strongest holdfast shedding phenotype. We determined that HfaB and HfaD are membrane-associated proteins and that HfaB is a lipoprotein. Purification of stalks and cell bodies indicated that both HfaB and HfaD are enriched in the stalk as compared to the cell body. These results suggest that HfaB and HfaD, and probably HfaA, serve to anchor the holdfast to the tip of the stalk.

Section: Phenotypic Analysis Of Hfa Mutantssupporting

confidence: 70%

Section: Detection Of the Holdfast With Fluorescein-labelled Lectinmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 95%

See 1 more Smart Citation

The HfaB and HfaD adhesion proteins of Caulobacter crescentus are localized in the stalk

Cole

Hardy

Bodenmiller

et al. 2003

Self Cite

“…Images were captured with a Princeton Instruments cooled CCD camera (model 1317) and METAMORPH imaging software from Universal Imaging Corporation (version 4.5). Lectin binding assays were performed as previously described (Janakiraman and Brun, 1999) and fluorescence observed using a Nikon B-2E FITC filter cube (Chroma Technologies). For fluorescent phage binding, a 6 ¥ 10 10 pfu ml -1 lysate of the Caulobacter phage fCbK was incubated at 4∞C for 48 h with the cyanine nucleic acid stain YO-PRO-1 (Molecular Probes, catalogue no.…”

Section: Microscopymentioning

confidence: 99%

The Caulobacter crescentus polar organelle development protein PodJ is differentially localized and is required for polar targeting of the PleC development regulator

Hinz

Larson

Smith

et al. 2003

Self Cite

105

181

Summary Regulation of polar development and cell division inCaulobacter crescentus relies on the dynamic localization of several proteins to cell poles at specific stages of the cell cycle. The p olar o rganelle d evelopment protein, PodJ, is required for the synthesis of the adhesive holdfast and pili. Here we show the cell cycle localization of PodJ and describe a novel role for this protein in controlling the dynamic localization of the developmental regulator PleC. In swarmer cells, a short form of PodJ is localized at the flagellated pole. Upon differentiation of the swarmer cell into a stalked cell, full length PodJ is synthesized and localizes to the pole opposite the stalk. In late predivisional cells, full length PodJ is processed into a short form which remains localized at the flagellar pole after cell division and is degraded during swarmer to stalked cell differentiation. Polar localization of the developmental regulator PleC requires the presence of PodJ. In contrast, the polar localization of PodJ is not dependent on the presence of PleC. These results indicate that PodJ is an important determinant for the localization of a major regulator of cell differentiation. Thus, PodJ acts directly or indirectly to target PleC to the incipient swarmer pole, to establish the cellular asymmetry that leads to the synthesis of holdfasts and pili at their proper subcellular location.

“…Images were captured with a Princeton Instruments cooled charge-coupled device camera and the METAMORPH imaging software package V.3. Transmission electron microscopy was performed as described previously (Janakiraman and Brun, 1999) using a Jeol JEM-1010 electron microscope at 60 kV. Cells were stained with 7.5% uranyl magnesium acetate for 5 min.…”

Section: Immunofluorescence and Microscopymentioning

confidence: 99%

A set of ftsZ mutants blocked at different stages of cell division in Caulobacter

Wang

Jones

Brun

2001

Self Cite

SummaryFtsZ is required throughout the cell division process in eubacteria and in archaea. We report the isolation of novel mutants of the FtsZ gene in Caulobacter crescentus. Clusters of charged amino acids were changed to alanine to minimize mutations that affect protein folding. Molecular modelling indicated that all the clustered-charged-to-alanine mutations had altered amino acids at the surface of the protein. Of 13 such mutants, four were recessive-lethal, three were dominant-lethal, and six had no discernible phenotype. An FtsZ depletion strain of Caulobacter was constructed to analyse the phenotype of the recessive-lethal mutations and used to show that they blocked cell division at distinct stages. One mutation blocked the initiation of cell division, two mutations blocked cell division randomly, and one mutation blocked both early and late stages of cell division. The effect of the recessive mutations on the subcellular localization of FtsZ was determined. Models to explain the various mutant phenotypes are discussed. This is the first set of recessive alleles of ftsZ blocked at different stages of cell division.