scholarly journals Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

1998 ◽  
Vol 180 (17) ◽  
pp. 4406-4412 ◽  
Author(s):  
Dominique Mengin-Lecreulx ◽  
Juan Ayala ◽  
Ahmed Bouhss ◽  
Jean van Heijenoort ◽  
Claudine Parquet ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Essential Gene ◽  
Cell Envelope ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Peptidoglycan Synthesis ◽  
Cell Wall Peptidoglycan ◽  
Expression Of Genes

ABSTRACT Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (P mra ) on the chromosome and its replacement by the lac promoter (P mra ::P lac ) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from P mra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which P mra ::P lac promoter expression was repressed or fully induced. It was confirmed that the P mra promoter is required for expression of the first nine genes of the mra cluster:mraZ (orfC), mraW(orfB), ftsL (mraR),ftsI, murE, murF, mraY,murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced P mra ::P lac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when P mra expression was repressed. It was concluded that the transcription of the genes located downstream fromftsW in the mra cluster, from murGto ftsZ, is also mainly (but not exclusively) dependent on the P mra promoter.

scholarly journals Role of Peptidoglycan Amidases in the Development and Morphology of the Division Septum in Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5334-5347 ◽  
Author(s):  
Richa Priyadarshini ◽  
Miguel A. de Pedro ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Envelope ◽  
Penicillin Binding Protein ◽  
Mature Cell ◽  
Daughter Cells ◽  
Peptidoglycan Synthesis ◽  
A Cell ◽  
Entire Cell

ABSTRACT Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.

scholarly journals Structural Determinants Required To Target Penicillin-Binding Protein 3 to the Septum of Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6110-6117 ◽  
Author(s):  
André Piette ◽  
Claudine Fraipont ◽  
Tanneke den Blaauwen ◽  
Mirjam E. G. Aarsman ◽  
Soumya Pastoret ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Penicillin Binding Protein ◽  
Structural Determinants ◽  
Membrane Anchor ◽  
Interaction Sites ◽  
Division Site ◽  
Green Fluorescent

ABSTRACT In Escherichia coli, cell division is mediated by the concerted action of about 12 proteins that assemble at the division site to presumably form a complex called the divisome. Among these essential division proteins, the multimodular class B penicillin-binding protein 3 (PBP3), which is specifically involved in septal peptidoglycan synthesis, consists of a short intracellular M1-R23 peptide fused to a F24-L39 membrane anchor that is linked via a G40-S70 peptide to an R71-I236 noncatalytic module itself linked to a D237-V577 catalytic penicillin-binding module. On the basis of localization analyses of PBP3 mutants fused to green fluorescent protein by fluorescence microscopy, it appears that the first 56 amino acid residues of PBP3 containing the membrane anchor and the G40-E56 peptide contain the structural determinants required to target the protein to the cell division site and that none of the putative protein interaction sites present in the noncatalytic module are essential for the positioning of the protein to the division site. Based on the effects of increasing production of FtsQ or FtsW on the division of cells expressing PBP3 mutants, it is suggested that these proteins could interact. We postulate that FtsQ could play a role in regulating the assembly of these division proteins at the division site and the activity of the peptidoglycan assembly machineries within the divisome.

scholarly journals Role of the Escherichia coli SurA Protein in Stationary-Phase Survival

1998 ◽  
Vol 180 (21) ◽  
pp. 5704-5711 ◽  
Author(s):  
Sara W. Lazar ◽  
Marta Almirón ◽  
Antonio Tormo ◽  
Roberto Kolter
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Stationary Phase ◽  
Binding Protein ◽  
Luria Bertani ◽  
Penicillin Binding Protein ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
Stationary Phase Survival

ABSTRACT SurA is a periplasmic peptidyl-prolyl isomerase required for the efficient folding of extracytoplasmic proteins. Although thesurA gene had been identified in a screen for mutants that failed to survive in stationary phase, the role played by SurA in stationary-phase survival remained unknown. The results presented here demonstrate that the survival defect of surA mutants is due to their inability to grow at elevated pH in the absence of ςS. When cultures of Escherichia coli were grown in peptide-rich Luria-Bertani medium, the majority of the cells lost viability during the first two to three days of incubation in stationary phase as the pH rose to pH 9. At this time the surviving cells resumed growth. In cultures of surA rpoS double mutants the survivors lysed as they attempted to resume growth at the elevated pH. Cells lacking penicillin binding protein 3 and ςS had a survival defect similar to that of surA rpoS double mutants, suggesting that SurA foldase activity is important for the proper assembly of the cell wall-synthesizing apparatus.

scholarly journals FtsI and FtsW Are Localized to the Septum inEscherichia coli

1998 ◽  
Vol 180 (11) ◽  
pp. 2810-2816 ◽  
Author(s):  
Lilin Wang ◽  
Medhat K. Khattar ◽  
W. D. Donachie ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Immunofluorescence Microscopy ◽  
Binding Protein ◽  
Specific Inhibitor ◽  
Inescherichia Coli ◽  
Penicillin Binding Protein ◽  
Cell Division Protein

ABSTRACT The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed inftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

scholarly journals Selected Amplification of the Cell Division Genes ftsQ-ftsA-ftsZ in Escherichia coli

Genetics ◽  
2000 ◽  
Vol 156 (4) ◽  
pp. 1483-1492
Author(s):  
Daniel Vinella ◽  
Michael Cashel ◽  
Richard D’Ari
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Insertional Mutagenesis ◽  
Penicillin Binding Protein ◽  
Insertional Inactivation ◽  
Ts Mutant ◽  
Direct Target ◽  
Resistant Mutants ◽  
General Method

Abstract Rapidly growing Escherichia coli is unable to divide in the presence of the antibiotic mecillinam, whose direct target is penicillin-binding protein 2 (PBP2), responsible for the elongation of the cylindrical portion of the cell wall. Division can be restored in the absence of PBP2 activity by increasing the concentration of the cell division proteins FtsQ, FtsA, and FtsZ. We tried to identify regulators of the ftsQ-ftsA-ftsZ operon among mecillinam-resistant mutants, which include strains overexpressing these genes. By insertional mutagenesis with mini-Tn10 elements, we selected for insertions that conferred mecillinam resistance. Among 15 such mutants, 7 suppressed the thermosensitivity of the ftsZ84(Ts) mutant, strongly suggesting that they had increased FtsZ activity. In all 7 cases, however, the mutants resulted from a duplication of the ftsQAZ region. These duplications seemed to result from multiple events, suggesting that no simple insertional inactivation can result in a mutant with sufficiently amplified ftsQAZ expression to confer mecillinam resistance. The structure of the duplications suggests a general method for constructing directed duplications of precise sequences.

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