Limits...
Screen for localized proteins in Caulobacter crescentus.

Russell JH, Keiler KC - PLoS ONE (2008)

Bottom Line: Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known.The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria.Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species.

Show MeSH

Related in: MedlinePlus

Localization of CC2233 changes during the cell cycle.Swarmer cells were isolated from cultures of C. crescentus producing CC2233(1–62)-GFP and the cells were imaged by epifluorescence (left panels) and DIC microscopy (right panels) during synchronous growth in M2G medium. The time after synchronization is shown (cell cycle progression is slower than in Figure 4 because cells were grown in minimal medium). Schematic diagram indicates the cell cycle stage at each time point. CC2233-GFP was located around the periphery until the predivisional stage (105–135 min), when it was concentrated at the division plane (arrowheads). Scale bars indicating 1 µm are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2262157&req=5

pone-0001756-g009: Localization of CC2233 changes during the cell cycle.Swarmer cells were isolated from cultures of C. crescentus producing CC2233(1–62)-GFP and the cells were imaged by epifluorescence (left panels) and DIC microscopy (right panels) during synchronous growth in M2G medium. The time after synchronization is shown (cell cycle progression is slower than in Figure 4 because cells were grown in minimal medium). Schematic diagram indicates the cell cycle stage at each time point. CC2233-GFP was located around the periphery until the predivisional stage (105–135 min), when it was concentrated at the division plane (arrowheads). Scale bars indicating 1 µm are shown.

Mentions: The transposon insertion in CC2233 resulted in a fusion protein containing only 62 amino acids of CC2233, which was localized to the periphery of the cells in 100% of cells (Fig. 8). Bioinformatic predictions did not suggest that CC2233 is a membrane protein, so to ensure that the full-length protein was also localized, the full-length CC2233 gene fused to gfp was cloned under the control of a vanillate-inducible promoter and expressed in C. crescentus. The fluorescence patterns in these cells were indistinguishable from those in the transposon-generated fusion (Fig. 9). Cells producing CC2233-GFP were synchronized and observed to determine if the localization pattern changed as a function of the cell cycle. In swarmer and stalked cells, the fluorescence signal was localized around the periphery of the cell, but in pre-divisional cells the majority of the fluorescence moved to the division plane (Fig. 9). This localization pattern suggests that CC2233 might play some role in the late processes of cell division. However, deletion of CC2233 did not result in any defects in cell division, morphology, or cell growth. It is possible that CC2233 is localized to the division plane through interactions with a cell division protein, but CC2233 itself is not important for cytokinesis. Alternatively, CC2233 may be involved in a non-essential process at the division plane. The function of both CC0572 and CC2233 remain to be established, but their localization patterns suggest that many proteins with complex and dynamic localization patterns remain to be identified.


Screen for localized proteins in Caulobacter crescentus.

Russell JH, Keiler KC - PLoS ONE (2008)

Localization of CC2233 changes during the cell cycle.Swarmer cells were isolated from cultures of C. crescentus producing CC2233(1–62)-GFP and the cells were imaged by epifluorescence (left panels) and DIC microscopy (right panels) during synchronous growth in M2G medium. The time after synchronization is shown (cell cycle progression is slower than in Figure 4 because cells were grown in minimal medium). Schematic diagram indicates the cell cycle stage at each time point. CC2233-GFP was located around the periphery until the predivisional stage (105–135 min), when it was concentrated at the division plane (arrowheads). Scale bars indicating 1 µm are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2262157&req=5

pone-0001756-g009: Localization of CC2233 changes during the cell cycle.Swarmer cells were isolated from cultures of C. crescentus producing CC2233(1–62)-GFP and the cells were imaged by epifluorescence (left panels) and DIC microscopy (right panels) during synchronous growth in M2G medium. The time after synchronization is shown (cell cycle progression is slower than in Figure 4 because cells were grown in minimal medium). Schematic diagram indicates the cell cycle stage at each time point. CC2233-GFP was located around the periphery until the predivisional stage (105–135 min), when it was concentrated at the division plane (arrowheads). Scale bars indicating 1 µm are shown.
Mentions: The transposon insertion in CC2233 resulted in a fusion protein containing only 62 amino acids of CC2233, which was localized to the periphery of the cells in 100% of cells (Fig. 8). Bioinformatic predictions did not suggest that CC2233 is a membrane protein, so to ensure that the full-length protein was also localized, the full-length CC2233 gene fused to gfp was cloned under the control of a vanillate-inducible promoter and expressed in C. crescentus. The fluorescence patterns in these cells were indistinguishable from those in the transposon-generated fusion (Fig. 9). Cells producing CC2233-GFP were synchronized and observed to determine if the localization pattern changed as a function of the cell cycle. In swarmer and stalked cells, the fluorescence signal was localized around the periphery of the cell, but in pre-divisional cells the majority of the fluorescence moved to the division plane (Fig. 9). This localization pattern suggests that CC2233 might play some role in the late processes of cell division. However, deletion of CC2233 did not result in any defects in cell division, morphology, or cell growth. It is possible that CC2233 is localized to the division plane through interactions with a cell division protein, but CC2233 itself is not important for cytokinesis. Alternatively, CC2233 may be involved in a non-essential process at the division plane. The function of both CC0572 and CC2233 remain to be established, but their localization patterns suggest that many proteins with complex and dynamic localization patterns remain to be identified.

Bottom Line: Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known.The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria.Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.

ABSTRACT
Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species.

Show MeSH
Related in: MedlinePlus