Effect of 5-bromouracil on the growth of a thymine auxotroph of Escherichia coli K-12

1971 ◽  
Vol 17 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Roosevelt J. Jones ◽  
Roger R. Hewitt
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Solid Medium ◽  
Cell Number ◽  
Bacteriostatic Effect ◽  
E Coli ◽  
Static Phase ◽  
Two Phases ◽  
K 12 ◽  
Mass Increase

An atypical viability response to 5-bromouracil has been observed in a thymine auxotroph of E. coli K-12. The response occurs in two phases, the first reflecting tolerance to the analogue during continued exponential growth and cell division. The second is a static phase during which viable number remains constant, while cell number and mass increase at a diminishing rate.During the latter phase filamentous cells increase in number and length. Examination of the cloning potential of cells after 10 h of growth in 5-bromouracil indicated that filamentous cells continue extension on solid medium into non-septate coils that are sterile. Other cells, presumably static when plated, readily form microcolonies free of defective members.Observed responses to penicillin, potentially stabilizing media, or added thymine suggest that 5-bromouracil evokes a bimodal response in this strain. The analogue exerts a bacteriostatic effect on some cells which remain viable for several hours. The bacteriocidal effect, presumably on cells continuing growth, interferes with cell division by preventing septation.

scholarly journals A Predicted ABC Transporter, FtsEX, Is Needed for Cell Division in Escherichia coli

2004 ◽  
Vol 186 (3) ◽  
pp. 785-793 ◽  
Author(s):  
Kari L. Schmidt ◽  
Nicholas D. Peterson ◽  
Ryan J. Kustusch ◽  
Mark C. Wissel ◽  
Becky Graham ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Abc Transporter ◽  
Luria Bertani ◽  
Wild Type ◽  
E Coli ◽  
Division Site ◽  
Site Localization ◽  
Free Media ◽  
Mass Increase

ABSTRACT FtsE and FtsX have homology to the ABC transporter superfamily of proteins and appear to be widely conserved among bacteria. Early work implicated FtsEX in cell division in Escherichia coli, but this was subsequently challenged, in part because the division defects in ftsEX mutants are often salt remedial. Strain RG60 has an ftsE::kan null mutation that is polar onto ftsX. RG60 is mildly filamentous when grown in standard Luria-Bertani medium (LB), which contains 1% NaCl, but upon shift to LB with no NaCl growth and division stop. We found that FtsN localizes to potential division sites, albeit poorly, in RG60 grown in LB with 1% NaCl. We also found that in wild-type E. coli both FtsE and FtsX localize to the division site. Localization of FtsX was studied in detail and appeared to require FtsZ, FtsA, and ZipA, but not the downstream division proteins FtsK, FtsQ, FtsL, and FtsI. Consistent with this, in media lacking salt, FtsA and ZipA localized independently of FtsEX, but the downstream proteins did not. Finally, in the absence of salt, cells depleted of FtsEX stopped dividing before any change in growth rate (mass increase) was apparent. We conclude that FtsEX participates directly in the process of cell division and is important for assembly or stability of the septal ring, especially in salt-free media.

scholarly journals Unstable Escherichia coli L Forms Revisited: Growth Requires Peptidoglycan Synthesis

2007 ◽  
Vol 189 (18) ◽  
pp. 6512-6520 ◽  
Author(s):  
Danièle Joseleau-Petit ◽  
Jean-Claude Liébart ◽  
Juan A. Ayala ◽  
Richard D'Ari
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
High Performance ◽  
Specific Inhibitor ◽  
Hypertonic Medium ◽  
Penicillin Binding Proteins ◽  
E Coli ◽  
Chromatography Analysis ◽  
Peptidoglycan Synthesis ◽  
K 12

ABSTRACT Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the β-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the β-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli—and possibly all L forms—have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.

scholarly journals Regulation of Cell Division, Biofilm Formation, and Virulence by FlhC in Escherichia coli O157:H7 Grown on Meat

2011 ◽  
Vol 77 (11) ◽  
pp. 3653-3662 ◽  
Author(s):  
Preeti Sule ◽  
Shelley M. Horne ◽  
Catherine M. Logue ◽  
Birgit M. Prüß
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Real Time ◽  
Real Time Pcr ◽  
Biofilm Formation ◽  
Parent Strain ◽  
Cellular Functions ◽  
Content Type ◽  
E Coli ◽  
K 12

ABSTRACTTo understand the continuous problems thatEscherichia coliO157:H7 causes as food pathogen, this study assessed global gene regulation in bacteria growing on meat. Since FlhD/FlhC ofE. coliK-12 laboratory strains was previously established as a major control point in transducing signals from the environment to several cellular processes, this study compared the expression pattern of anE. coliO157:H7 parent strain to that of its isogenicflhCmutant. This was done with bacteria that had been grown on meat. Microarray experiments revealed 287 putative targets of FlhC. Real-time PCR was performed as an alternative estimate of transcription and confirmed microarray data for 13 out of 15 genes tested (87%). The confirmed genes are representative of cellular functions, such as central metabolism, cell division, biofilm formation, and pathogenicity. An additional 13 genes from the same cellular functions that had not been hypothesized as being regulated by FlhC by the microarray experiment were tested with real-time PCR and also exhibited higher expression levels in theflhCmutant than in the parent strain. Physiological experiments were performed and confirmed that FlhC reduced the cell division rate, the amount of biofilm biomass, and pathogenicity in a chicken embryo lethality model. Altogether, this study provides valuable insight into the complex regulatory network of the pathogen that enables its survival under various environmental conditions. This information may be used to develop strategies that could be used to reduce the number of cells or pathogenicity ofE. coliO157:H7 on meat by interfering with the signal transduction pathways.

scholarly journals Deficiency in l-Serine Deaminase Interferes with One-Carbon Metabolism and Cell Wall Synthesis in Escherichia coli K-12

2010 ◽  
Vol 192 (20) ◽  
pp. 5515-5525 ◽  
Author(s):  
Xiao Zhang ◽  
Ziad W. El-Hajj ◽  
Elaine Newman
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Division ◽  
Amino Acid ◽  
Cell Wall Synthesis ◽  
E Coli ◽  
Amino Acid Mixtures ◽  
Glycine Cleavage ◽  
High Level ◽  
K 12

ABSTRACT Escherichia coli K-12 provided with glucose and a mixture of amino acids depletes l-serine more quickly than any other amino acid even in the presence of ammonium sulfate. A mutant without three 4Fe4S l-serine deaminases (SdaA, SdaB, and TdcG) of E. coli K-12 is unable to do this. The high level of l-serine that accumulates when such a mutant is exposed to amino acid mixtures starves the cells for C1 units and interferes with cell wall synthesis. We suggest that at high concentrations, l-serine decreases synthesis of UDP-N-acetylmuramate-l-alanine by the murC-encoded ligase, weakening the cell wall and producing misshapen cells and lysis. The inhibition by high l-serine is overcome in several ways: by a large concentration of l-alanine, by overproducing MurC together with a low concentration of l-alanine, and by overproducing FtsW, thus promoting septal assembly and also by overexpression of the glycine cleavage operon. S-Adenosylmethionine reduces lysis and allows an extensive increase in biomass without improving cell division. This suggests that E. coli has a metabolic trigger for cell division. Without that reaction, if no other inhibition occurs, other metabolic functions can continue and cells can elongate and replicate their DNA, reaching at least 180 times their usual length, but cannot divide.

scholarly journals Structural genes for nitrate-inducible formate dehydrogenase in Escherichia coli K-12.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 691-702 ◽  
Author(s):  
B L Berg ◽  
V Stewart
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Formate Dehydrogenase ◽  
Recombinant Dna ◽  
Structural Genes ◽  
Respiratory Pathway ◽  
E Coli ◽  
Formate Oxidation ◽  
Operon Expression ◽  
K 12

Abstract Formate oxidation coupled to nitrate reduction constitutes a major anaerobic respiratory pathway in Escherichia coli. This respiratory chain consists of formate dehydrogenase-N, quinone, and nitrate reductase. We have isolated a recombinant DNA clone that likely contains the structural genes, fdnGHI, for the three subunits of formate dehydrogenase-N. The fdnGHI clone produced proteins of 110, 32 and 20 kDa which correspond to the subunit sizes of purified formate dehydrogenase-N. Our analysis indicates that fdnGHI is organized as an operon. We mapped the fdn operon to 32 min on the E. coli genetic map, close to the genes for cryptic nitrate reductase (encoded by the narZ operon). Expression of phi(fdnG-lacZ) operon fusions was induced by anaerobiosis and nitrate. This induction required fnr+ and narL+, two regulatory genes whose products are also required for the anaerobic, nitrate-inducible activation of the nitrate reductase structural gene operon, narGHJI. We conclude that regulation of fdnGHI and narGHJI expression is mediated through common pathways.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

Potentiation by L-cysteine of the bactericidal effect of hydrogen peroxide in Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 81-88
Author(s):  
E H Berglin ◽  
M B Edlund ◽  
G K Nyberg ◽  
J Carlsson
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Metal Ion ◽  
Chelating Agent ◽  
Fold Increase ◽  
Bactericidal Effect ◽  
Anaerobic Conditions ◽  
Dna Breakage ◽  
E Coli ◽  
K 12

Under anaerobic conditions an exponentially growing culture of Escherichia coli K-12 was exposed to hydrogen peroxide in the presence of various compounds. Hydrogen peroxide (0.1 mM) together with 0.1 mM L-cysteine or L-cystine killed the organisms more rapidly than 10 mM hydrogen peroxide alone. The exposure of E. coli to hydrogen peroxide in the presence of L-cysteine inhibited some of the catalase. This inhibition, however, could not fully explain the 100-fold increase in hydrogen peroxide sensitivity of the organism in the presence of L-cysteine. Of other compounds tested only some thiols potentiated the bactericidal effect of hydrogen peroxide. These thiols were effective, however, only at concentrations significantly higher than 0.1 mM. The effect of L-cysteine and L-cystine could be annihilated by the metal ion chelating agent 2,2'-bipyridyl. DNA breakage in E. coli K-12 was demonstrated under conditions where the organisms were killed by hydrogen peroxide.

scholarly journals pH-Dependent Catabolic Protein Expression during Anaerobic Growth of Escherichia coli K-12

2004 ◽  
Vol 186 (1) ◽  
pp. 192-199 ◽  
Author(s):  
Elizabeth Yohannes ◽  
D. Michael Barnhart ◽  
Joan L. Slonczewski
Keyword(s):  
Escherichia Coli ◽  
Ph Regulation ◽  
Metabolic Enzymes ◽  
Anaerobic Growth ◽  
High Ph ◽  
E Coli ◽  
Catabolic Enzymes ◽  
Periplasmic Proteins ◽  
Ph Dependent ◽  
K 12

ABSTRACT During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.

scholarly journals The Small Noncoding DsrA RNA Is an Acid Resistance Regulator in Escherichia coli

2004 ◽  
Vol 186 (18) ◽  
pp. 6179-6185 ◽  
Author(s):  
Richard A. Lease ◽  
Dorie Smith ◽  
Kathleen McDonough ◽  
Marlene Belfort
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Resistance Genes ◽  
Acid Resistance ◽  
Dna Arrays ◽  
E Coli ◽  
Specific Mrnas ◽  
Steady State Levels ◽  
Control Translation ◽  
K 12

ABSTRACT DsrA RNA is a small (87-nucleotide) regulatory RNA of Escherichia coli that acts by RNA-RNA interactions to control translation and turnover of specific mRNAs. Two targets of DsrA regulation are RpoS, the stationary-phase and stress response sigma factor (σs), and H-NS, a histone-like nucleoid protein and global transcription repressor. Genes regulated globally by RpoS and H-NS include stress response proteins and virulence factors for pathogenic E. coli. Here, by using transcription profiling via DNA arrays, we have identified genes induced by DsrA. Steady-state levels of mRNAs from many genes increased with DsrA overproduction, including multiple acid resistance genes of E. coli. Quantitative primer extension analysis verified the induction of individual acid resistance genes in the hdeAB, gadAX, and gadBC operons. E. coli K-12 strains, as well as pathogenic E. coli O157:H7, exhibited compromised acid resistance in dsrA mutants. Conversely, overproduction of DsrA from a plasmid rendered the acid-sensitive dsrA mutant extremely acid resistant. Thus, DsrA RNA plays a regulatory role in acid resistance. Whether DsrA targets acid resistance genes directly by base pairing or indirectly via perturbation of RpoS and/or H-NS is not known, but in either event, our results suggest that DsrA RNA may enhance the virulence of pathogenic E. coli.

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