ftsZ is an essential cell division gene in Escherichia coli.

The ftsZ gene is thought to be an essential cell division gene in Escherichia coli. We constructed a null allele of ftsZ in a strain carrying additional copies of ftsZ on a plasmid with a temperature-sensitive replication defect. This strain was temperature sensitive for cell division and viability, confirming that ftsZ is an essential cell division gene. Further analysis revealed that after a shift to the nonpermissive temperature, cell division ceased when the level of FtsZ started to decrease, indicating that septation is very sensitive to the level of FtsZ. Subsequent studies showed that nucleoid segregation was normal while FtsZ was decreasing and that ftsZ expression was not autoregulated. The null allele could not be complemented by lambda 16-2, even though this bacteriophage can complement the thermosensitive ftsZ84 mutation and carries 6 kb of DNA upstream of the ftsZ gene.     

yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli

The outer membrane of Gram‐negative bacteria is an asymmetric lipid bilayer consisting of an essential glycolipid lipopolysaccharide (LPS) in its outer leaflet and phospholipids in the inner leaflet. Here, we show that yciM, a gene encoding a tetratricopeptide repeat protein of unknown function, modulates LPS levels by negatively regulating the biosynthesis of lipid A, an essential constituent of LPS. Inactivation of yciM resulted in high LPS levels and cell death in Escherichia coli; recessive mutations in lpxA, lpxC or lpxD that lower the synthesis of lipid A, or a gain of function mutation in fabZ that increases the formation of membrane phospholipids, alleviated the yciM mutant phenotypes. A modest increase in YciM led to significant reduction of LPS and increased sensitivity to hydrophobic antibiotics. YciM was shown to regulate LPS by altering LpxC, an enzyme that catalyses the first committed step of lipid A biosynthesis. Regulation of LpxC by YciM was contingent on the presence of FtsH, an essential membrane‐anchored protease known to degrade LpxC, suggesting that FtsH and YciM act in concert to regulate synthesis of lipid A. In summary, this study demonstrates an essential role for YciM in regulation of LPS biosynthesis in E. coli.     

SigmaE is an essential sigma factor in Escherichia coli.

SigmaE is an alternative sigma factor that controls the extracytoplasmic stress response in Escherichia coli. SigmaE is essential at high temperatures but was previously thought to be nonessential at temperatures below 37 degrees C. We present evidence that sigmaE is an essential sigma factor at all temperatures. Cells lacking sigmaE are able to grow at low temperatures because of the presence of a frequently arising, unlinked suppressor mutation.     

The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

The murI gene of Escherichia coli was recently identified on the basis of its ability to complement the only mutant requiring D-glutamic acid for growth that had been described to date: strain WM335 of E. coli B/r (P. Doublet, J. van Heijenoort, and D. Mengin-Lecreulx, J. Bacteriol. 174:5772-5779, 1992). We report experiments of insertional mutagenesis of the murI gene which demonstrate that this gene is essential for the biosynthesis of D-glutamic acid, one of the specific components of cell wall peptidoglycan. A special strategy was used for the construction of strains with a disrupted copy of murI, because of a limited capability of E. coli strains grown in rich medium to internalize D-glutamic acid. The murI gene product was overproduced and identified as a glutamate racemase activity. UDP-N-acetylmuramoyl-L-alanine (UDP-MurNAc-L-Ala), which is the nucleotide substrate of the D-glutamic-acid-adding enzyme (the murD gene product) catalyzing the subsequent step in the pathway for peptidoglycan synthesis, appears to be an effector of the racemase activity.     

Methionine aminopeptidase gene of Escherichia coli is essential for cell growth.

We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4. Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-beta-thiogalactoside. Thus, methionine aminopeptidase is essential for cell growth.     

Early stages in development of the Escherichia coli cell-division site

Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ^- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ^- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.     

surA, an Escherichia coli gene essential for survival in stationary phase.

Mutations in genes not required for exponential growth but essential for survival in stationary phase were isolated in an effort to understand the ability of wild-type Escherichia coli cells to remain viable during prolonged periods of nutritional deprivation. The phenotype of these mutations is referred to as Sur- (survival) and the genes are designated sur. The detailed analysis of one of these mutations is presented here. The mutation (surA1) caused by insertion of a mini-Tn10 element defined a new gene located near 1 min on the E. coli chromosome. It was located directly upstream of pdxA and formed part of a complex operon. Evidence is presented supporting the interpretation that cells harboring the surA1 mutation die during stationary phase while similar insertion mutations in other genes of the operon do not lead to a Sur- phenotype. Strains harboring surA1 had a normal doubling time in both rich and minimal medium, but cultures lost viability after several days in stationary phase. Analysis of revertants and suppressors of surA1, which arose after prolonged incubation in stationary phase, indicates that DNA rearrangements (excisions and duplications) occurred in cultures of this strain even when the viable-cell counts were below 10(2) cells per ml. Cells containing suppressing mutations then grew in the same culture to 10(8) cells per ml, taking over the population. The implications of these observations to our understanding of stationary-phase mutagenesis are discussed.     

The 4.5 S RNA gene of Escherichia coli is essential for cell growth

The Escherichia coli gene coding for the metabolically stable 4.5 S RNA (ffs) has been shown to be required for cell viability. Essentiality was demonstrated by examining the recombination behavior of substitution mutations of ffs generated in vitro. Substitution mutants of ffs are able to replace the chromosomal allele only in the presence of a second, intact copy of ffs. Independent evidence of essentiality and the finding that 4.5 S RNA is important for protein synthetic activity came from characterization of cells dependent on the lac operon inducer isopropyl-beta-D-thiogalactoside for ffs gene expression. Here, a strain dependent on isopropyl-beta-D-thiogalactoside for 4.5 S RNA synthesis was developed by inactivation of the chromosomal ffs allele and lysogenization by a lambda phage containing 4.5 S DNA fused to a hybrid trp-lac promoter. Withdrawal of the thiogalactoside leads to a deficiency in 4.5 S RNA, a dramatic loss in protein synthesis activity, and eventual cell death. Tagging of the chromosomal ffs region with a kanamycin-resistance gene allowed mapping of the 4.5 S RNA gene. Results from this analysis place ffs near lon at approximately ten minutes on the E. coli linkage map.     

Involvement of an essential gene, mviN, in murein synthesis in Escherichia coli

We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.     

MioC is an FMN-binding protein that is essential for Escherichia coli biotin synthase activity in vitro

Biotin synthase is required for the conversion of dethiobiotin to biotin and requires a number of accessory proteins and small molecule cofactors for activity in vitro. We have previously identified two of these proteins as flavodoxin and ferredoxin (flavodoxin) NADP(+) reductase. We now report the identification of MioC as a third essential protein, together with its cloning, purification, and characterization. Purified MioC has a UV-visible spectrum characteristic of a flavoprotein and contains flavin mononucleotide. The presence of flavin mononucleotide and the primary sequence similarity to flavodoxin suggest that MioC may function as an electron transport protein. The role of MioC in the biotin synthase reaction is discussed, and the structure and function of MioC is compared with that of flavodoxin.     

Switching control of an essential gene for reprogramming of cellular phenotypes in Escherichia coli

Biological systems designs require various dynamic controllers capable of modulating cellular phenotypes to adapt to changing environments. Cellular phenotypes are simultaneously affected by combinations of multiple genes that are controlled by global regulators. However, it is difficult to intentionally control the expression of these global regulators dynamically because they are essential for cell survival and are involved in regulatory networks clustered in operons. Here, we designed a platform that allows dynamic modulation of the expression of an essential gene. Using this system, comprising of on/off switches that respond to an extracellular stimulus, we successfully demonstrated the switching control of the expression of fusA encoding elongation factor G (EF-G). An additional control module in this system that responds to changed external signals was shown to provide the capacity to "switch gears" and reprogram cellular phenotypes with desired timing.     

Nuclease activity is essential for RecBCD recombination in Escherichia coli

RecBCD has two conflicting roles in Escherichia coli. (i) As ExoV, it is a potent double-stranded (ds)DNA exonuclease that destroys linear DNA produced by restriction of foreign DNA. (ii) As a recombinase, it promotes repair of dsDNA breaks and genetic recombination in the vicinity of chi recombination hot-spots. These paradoxical roles are accommodated by chi-dependent attenuation of RecBCD exonuclease activity and concomitant conversion of the enzyme to a recombinase. To challenge the proposal that chi converts RecBCD from a destructive exonuclease to a recombinogenic helicase, we mutated the nuclease catalytic centre of RecB and tested the resulting mutants for genetic recombination and DNA repair in vivo. We predicted that, if nuclease activity inhibits recombination and helicase activity is sufficient for recombination, the mutants would be constitutive recombinases, as has been seen in recD null mutants. Conversely, if nuclease activity is required, the mutants would be recombination deficient. Our results indicate that 5' --> 3' exonuclease activity is essential for recombination by RecBCD at chi recombination hot-spots and at dsDNA ends in recD mutants. In the absence of RecB-dependent nuclease function, recombination becomes entirely dependent on the 5' --> 3' single-stranded (ss)DNA exonuclease activity of RecJ and the helicase activity of RecBC(D).     

Pyrophosphatase is essential for growth of Escherichia coli.

The ppa gene for inorganic pyrophosphatase is essential for the growth of Escherichia coli. A recombinant with a chromosomal ppa::Kanr lesion and a temperature-sensitive replicon with a ppa+ gene showed a temperature-sensitive growth phenotype, and a mutant with the sole ppa+ gene under control of the lac promoter showed inducer-dependent growth. When the lacp-ppa mutant was subcultured without inducer, the pyrophosphatase level decreased, the PPi level increased, and growth stopped. Cellular PPi reached 16 mM about 6 h after growth arrest without loss of cell viability.     

Method for determining whether a gene of Escherichia coli is essential: application to the polA gene

We have developed a general method for determining whether a gene of Escherichia coli is essential for viability. The method requires cloned DNA spanning the gene in question and a reasonably detailed genetic and physical map of the cloned segment. Using this information, one constructs a deletion of the target gene in vitro. For convenience, the deletion can be marked by an antibiotic resistance gene. A DNA segment containing the deletion is then cloned onto an att delta phage lambda vector. Integration of this phage, by homologous recombination at the target locus, and subsequent excision provide an efficient route for crossing the marked deletion onto the bacterial chromosome. Failure to delete the target gene indicates either that the resulting deletion was not viable or that the desired recombinational event did not take place. The use of prophage excision to generate the deletion allows one to estimate the fraction of deletion-producing events by analysis of the other product of the excision, the phage produced on induction of the prophage. In this way one can determine whether failure to recover a particular chromosomal deletion was due to its never having been formed, or, once formed, to its failure to survive. Applying this method to the polA gene, we found that polA is required for growth on rich medium but not on minimal medium. We repeated the experiment in the presence of plasmids carrying functional fragments of the polA gene, corresponding to the 5'-3' exonuclease and the polymerase-3'-5' exonuclease portions of DNA polymerase I. Surprisingly, either of these fragments, in the absence of the other, was sufficient to allow growth on rich medium.     

Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.     

The ispB gene encoding octaprenyl diphosphate synthase is essential for growth of Escherichia coli.

The Escherichia coli ispB gene encoding octaprenyl diphosphate synthase is responsible for the synthesis of the side chain of isoprenoid quinones. We tried to construct an E. coli ispB-disrupted mutant but could not isolate the chromosomal ispB disrupted mutant unless the ispB gene or its homolog was supplied on a plasmid. The chromosomal ispB disruptants that harbored plasmids carrying the ispB homologs from Haemophilus influenzae and Synechocystis sp. strain PCC6803 produced mainly ubiquinone 7 and ubiquinone 9, respectively. Our results indicate that the function of the ispB gene is essential for normal growth and that this function can be substituted for by homologs of the ispB gene from other organisms that produce distinct forms of ubiquinone.     

Iron uptake is essential for Escherichia coli survival in drinking water

AIMS: The aim of this study was to elucidate if the need for iron for Escherichia coli to remain cultivable in a poorly nutritive medium such as the drinking water uses the iron transport system via the siderophores. METHODS AND RESULTS: Environmental strains of E. coli (isolated from a drinking water network), referenced strains of E. coli and mutants deficient in TonB, an essential protein for iron(III) acquisition, were incubated for 3 weeks at 25 degrees C, in sterile drinking water with and without lepidocrocite (gamma-FeOOH), an insoluble iron corrosion product. Only cells with a functional iron transport system were able to survive throughout the weeks. CONCLUSIONS: The iron transport system via protein TonB plays an essential role on the survival of E. coli in a weakly nutritive medium like drinking water. SIGNIFICANCE AND IMPACTS OF THE STUDY: Iron is a key parameter involved in coliform persistence in drinking water distribution systems.