Temperature-sensitive sec mutants of Escherichia coli: inhibition of protein export at the permissive temperature.

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

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The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

Biochemical Journal ◽

10.1042/bj1400443 ◽

1974 ◽

Vol 140 (3) ◽

pp. 443-450 ◽

Cited By ~ 2

Author(s):

John R. Dean ◽

John Sykes

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Maximum Activity ◽

Exponential Phase ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Experimental Conditions ◽

Inhibition Of Growth ◽

Ribosomal Ribonucleic Acid

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

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fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli

Microbiology ◽

10.1099/mic.0.26337-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2427-2433 ◽

Cited By ~ 6

Author(s):

Martin Stancek ◽

Leif A. Isaksson ◽

Monica Rydén-Aulin

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Temperature Sensitivity ◽

Minimal Medium ◽

Synthetic Medium ◽

Normal Growth ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Atp Binding Site ◽

Pleiotropic Phenotype

Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.

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Functional Analysis of the Signal Recognition Particle in Escherichia coli by Characterization of a Temperature-Sensitive ffh Mutant

Journal of Bacteriology ◽

10.1128/jb.184.10.2642-2653.2002 ◽

2002 ◽

Vol 184 (10) ◽

pp. 2642-2653 ◽

Cited By ~ 20

Author(s):

Sei-Kyoung Park ◽

Fenglei Jiang ◽

Ross E. Dalbey ◽

Gregory J. Phillips

Keyword(s):

Escherichia Coli ◽

Signal Recognition Particle ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Signal Recognition ◽

E Coli ◽

Elevated Expression ◽

Type Gene ◽

Shock Proteins

ABSTRACT The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42°C as well as show reduced growth at the permissive temperature of 30°C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42°C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function.

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TEMPERATURE-SENSITIVE MUTANTS FOR THE REPLICATION OF PLASMIDS IN ESCHERICHIA COLI II. PROPERTIES OF HOST AND PLASMID MUTATIONS

Genetics ◽

10.1093/genetics/74.1.1 ◽

1973 ◽

Vol 74 (1) ◽

pp. 1-16

Author(s):

David T Kingsbury ◽

Donna G Sieckmann ◽

Donald R Helinski

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Cell Growth ◽

Dna Synthesis ◽

Plasmid Dna ◽

Recipient Cell ◽

Conjugal Transfer ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Group I

ABSTRACT Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E1 (ColE1) exhibit a pleiotropic effect with respect to the effect of the mutation on other extrachromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43°C required for complete cessation of COlE1, DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE1 DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE1 plasmid resulted in only a gradual cessation of ColE1 DNA synthesis over several generations of cell growth at 43°C. Conjugal transfer of the ColE1 and COlV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43°C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of COlE1 or COlV in the mutant recipient cell upon conjugation with a normal donor at 43°C. Various host COlE1, replication mutants carrying either ColE1 or ColE2 were also defective in the mitomycin Cinduced production of colicin E1 or colicin E2 at 43°C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.

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Components of the Salmonella Flagellar Export Apparatus and Classification of Export Substrates

Journal of Bacteriology ◽

10.1128/jb.181.5.1388-1394.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1388-1394 ◽

Cited By ~ 246

Author(s):

Tohru Minamino ◽

Robert M. Macnab

Keyword(s):

Outer Membrane ◽

Protein Export ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Capping Protein ◽

Ring Assembly ◽

Suitable Temperature ◽

Bona Fide ◽

Filament Type

ABSTRACT Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) required FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm. In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export. No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins. We suspect that FliJ may be a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook-type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.

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Generation of pre-tRNAs from polycistronic operons is the essential function of RNase P in Escherichia coli

Nucleic Acids Research ◽

10.1093/nar/gkz1188 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2564-2578 ◽

Cited By ~ 2

Author(s):

Bijoy K Mohanty ◽

Ankit Agrawal ◽

Sidney R Kushner

Keyword(s):

Escherichia Coli ◽

Rnase P ◽

Ribonuclease P ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Essential Function ◽

Sensitive Allele ◽

Polymerase I ◽

First Time

Abstract Ribonuclease P (RNase P) is essential for the 5′-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5′-immature tRNAs can be aminoacylated unless their 5′-extensions ≥8 nt. Surprisingly, the failure of 5′-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5′-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.

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New Temperature-Sensitive Alleles of ftsZ in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.187.1.358-365.2005 ◽

2005 ◽

Vol 187 (1) ◽

pp. 358-365 ◽

Cited By ~ 18

Author(s):

Stephen G. Addinall ◽

Elaine Small ◽

Duncan Whitaker ◽

Shane Sturrock ◽

William D. Donachie ◽

...

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Point Mutations ◽

Ring Formation ◽

Single Amino Acid ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Nonpermissive Temperature ◽

Z Ring

ABSTRACT We isolated five new temperature-sensitive alleles of the essential cell division gene ftsZ in Escherichia coli, using P1-mediated, localized mutagenesis. The five resulting single amino acid changes (Gly109→Ser109 for ftsZ6460, Ala129→Thr129 for ftsZ972, Val157→Met157 for ftsZ2066, Pro203→Leu203 for ftsZ9124, and Ala239→Val239 for ftsZ2863) are distributed throughout the FtsZ core region, and all confer a lethal cell division block at the nonpermissive temperature of 42°C. In each case the division block is associated with loss of Z-ring formation such that fewer than 2% of cells show Z rings at 42°C. The ftsZ9124 and ftsZ6460 mutations are of particular interest since both result in abnormal Z-ring formation at 30°C and therefore cause significant defects in FtsZ polymerization, even at the permissive temperature. Neither purified FtsZ9124 nor purified FtsZ6460 exhibited polymerization when it was assayed by light scattering or electron microscopy, even in the presence of calcium or DEAE-dextran. Hence, both mutations also cause defects in FtsZ polymerization in vitro. Interestingly, FtsZ9124 has detectable GTPase activity, although the activity is significantly reduced compared to that of the wild-type FtsZ protein. We demonstrate here that unlike expression of ftsZ84, multicopy expression of the ftsZ6460, ftsZ972, and ftsZ9124 alleles does not complement the respective lethalities at the nonpermissive temperature. In addition, all five new mutant FtsZ proteins are stable at 42°C. Therefore, the novel isolates carrying single ftsZ(Ts) point mutations, which are the only such strains obtained since isolation of the classical ftsZ84 mutation, offer significant opportunities for further genetic characterization of FtsZ and its role in cell division.

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The effects of acridine orange on deoxyribonucleic acid in Escherichia coli

Biochemical Journal ◽

10.1042/bj1710567 ◽

1978 ◽

Vol 171 (3) ◽

pp. 567-573 ◽

Cited By ~ 1

Author(s):

G R Barker ◽

N Hardman

Keyword(s):

Escherichia Coli ◽

Acridine Orange ◽

Dna Polymerase ◽

Deoxyribonucleic Acid ◽

Insoluble Fraction ◽

Low Molecular Weight ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Dna Polymerase I ◽

Polymerase I

1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.

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