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1.
Mol Microbiol ; 100(1): 1-14, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26593043

ABSTRACT

Bacterial morphology is determined primarily by the architecture of the peptidoglycan (PG) cell wall, a mesh-like layer that encases the cell. To identify novel mechanisms that create or maintain cell shape in Escherichia coli, we used flow cytometry to screen a transposon insertion library and identified a wecE mutant that altered cell shape, causing cells to filament and swell. WecE is a sugar aminotransferase involved in the biosynthesis of enterobacterial common antigen (ECA), a non-essential outer membrane glycolipid of the Enterobacteriaceae. Loss of wecE interrupts biosynthesis of ECA and causes the accumulation of the undecaprenyl pyrophosphate-linked intermediate ECA-lipid II. The wecE shape defects were reversed by: (i) preventing initiation of ECA biosynthesis, (ii) increasing the synthesis of the lipid carrier undecaprenyl phosphate (Und-P), (iii) diverting Und-P to PG synthesis or (iv) promoting Und-P recycling. The results argue that the buildup of ECA-lipid II sequesters part of the pool of Und-P, which, in turn, adversely affects PG synthesis. The data strongly suggest there is competition for a common pool of Und-P, whose proper distribution to alternate metabolic pathways is required to maintain normal cell shape in E. coli.


Subject(s)
Antigens, Bacterial/metabolism , Escherichia coli/physiology , Metabolic Networks and Pathways , Polyisoprenyl Phosphates/metabolism , Antigens, Bacterial/genetics , Cell Wall/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial , Genotype , Mutation , Transaminases/genetics , Transaminases/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/metabolism
2.
J Bacteriol ; 195(4): 855-66, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23243305

ABSTRACT

Bacterial morphology imparts physiological advantages to cells in different environments and, judging by the fidelity with which shape is passed to daughter cells, is a tightly regulated characteristic. Surprisingly, only in the past 10 to 15 years has significant headway been made in identifying the mechanisms by which cells create and maintain particular shapes. One reason for this is that the relevant discoveries have relied heavily on the arduous, somewhat subjective process of manual microscopy. Here, we show that flow cytometry, coupled with the sorting capability of fluorescence-activated cell sorting (FACS), can detect, quantify, and enrich bacteria with morphological alterations. The light scattering properties of several highly aberrant morphological mutants of Escherichia coli were characterized by flow cytometry. Cells from a region that overlapped the distribution of normal rod-shaped cells were collected by FACS and reincubated. After 4 to 15 iterations of this enrichment process, suppressor mutants were isolated that returned almost all the population to a near-normal shape. Suppressors were successfully isolated from strains lacking three or four penicillin binding proteins (PBPs) but not from a mutant lacking a total of seven PBPs. The peptidoglycan endopeptidase, AmpH, was identified as being important for the suppression process, as was a related endopeptidase, MepA. The results validate the use of cell sorting as a means for studying bacterial morphology and identify at least one new class of enzymes required for the suppression of cell shape defects.


Subject(s)
Endopeptidases/metabolism , Escherichia coli/cytology , Gene Expression Regulation, Bacterial/physiology , Gene Expression Regulation, Enzymologic/physiology , Peptidoglycan/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cell Separation , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Endopeptidases/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flow Cytometry , Mutation , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Peptidoglycan Glycosyltransferase/genetics , Peptidoglycan Glycosyltransferase/metabolism , Phenotype , Plasmids
3.
J Bacteriol ; 190(6): 2065-74, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18192383

ABSTRACT

Gram-negative bacteria possess stress responses to maintain the integrity of the cell envelope. Stress sensors monitor outer membrane permeability, envelope protein folding, and energization of the inner membrane. The systems used by gram-negative bacteria to sense and combat stress resulting from disruption of the peptidoglycan layer are not well characterized. The peptidoglycan layer is a single molecule that completely surrounds the cell and ensures its structural integrity. During cell growth, new peptidoglycan subunits are incorporated into the peptidoglycan layer by a series of enzymes called the penicillin-binding proteins (PBPs). To explore how gram-negative bacteria respond to peptidoglycan stress, global gene expression analysis was used to identify Escherichia coli stress responses activated following inhibition of specific PBPs by the beta-lactam antibiotics amdinocillin (mecillinam) and cefsulodin. Inhibition of PBPs with different roles in peptidoglycan synthesis has different consequences for cell morphology and viability, suggesting that not all perturbations to the peptidoglycan layer generate equivalent stresses. We demonstrate that inhibition of different PBPs resulted in both shared and unique stress responses. The regulation of capsular synthesis (Rcs) phosphorelay was activated by inhibition of all PBPs tested. Furthermore, we show that activation of the Rcs phosphorelay increased survival in the presence of these antibiotics, independently of capsule synthesis. Both activation of the phosphorelay and survival required signal transduction via the outer membrane lipoprotein RcsF and the response regulator RcsB. We propose that the Rcs pathway responds to peptidoglycan damage and contributes to the intrinsic resistance of E. coli to beta-lactam antibiotics.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Capsules/metabolism , Escherichia coli/metabolism , Peptidoglycan/metabolism , Amdinocillin/pharmacology , Cefsulodin/pharmacology , Drug Resistance, Microbial/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Models, Biological , Oligonucleotide Array Sequence Analysis , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Polymerase Chain Reaction , Receptors, sigma/genetics , Receptors, sigma/metabolism
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