A temperature-sensitive mutant ofEscherichia coli with an alteration in ribosomal protein L22

A temperature-sensitive, protein synthesis-defective mutant ofEscherichia coli exhibiting an altered ribosomal protein L22 has been investigated. The temperature-sensitive mutation was mapped to therplV gene for protein L22. The genes from the wild type and mutant strains were amplified by the polymerase chain reaction and the products were sequenced. A cytosine to thymine transition at position 22 of the coding sequence was found in the mutant DNA, predicting an arginine to cysteine alteration in the protein. A single cysteine residue was found in the isolated mutant protein. This amino acid change accounts for the altered mobility of the mutant protein in two-dimensional gels and during reversed-phase HPLC. The temperature-sensitive phenotype was fully complemented by a plasmid carrying the wild type L22 gene. Ribosomes from the complemented cells showed only wild type protein L22 by two dimensional gel analysis and were as heat-resistant as control ribosomes in a translation assay. The point mutation in the L22 gene is uniquely responsible for the temperature-sensitivity of this strain.     

Characterization of the autophosphorylation of Era, an essential Escherichia coli GTPase

Era is an essential protein in Escherichia coli which binds both GTP and GDP and has an intrinsic GTPase activity. Studies on the role of GTP/GDP binding and GTPase activity in an attempt to understand its function lead to the observation that Era is autophosphorylated. The autophosphorylated reaction is specific for GTP and cannot use ATP as a phosphoryl group donor. The reaction velocity is of first order with respect to protein concentration, suggesting an intramolecular mechanism. Autophosphorylation occurs at serine and threonine residues. The major phosphorylated tryptic peptide isolated after autophosphorylation has been identified as ISITSR, from residue 33 to 38. The peptide contains the site of phosphorylation and two potential sites for serine and threonine phosphorylation. Subsequently, both the threonine residue at position 36 and the serine residue at position 37 were altered to alanine. The double mutant Era, but not individual single mutants, was unable to functionally complement the growth of an E. coli strain which cannot produce wild-type Era protein at high temperature. This suggests that either threonine 36 or serine 37 has to exist for the function of Era In vivo. phosphorylation of Era was also examined by two-dimensional gel electrophoresis. Era has been previously assigned two distinct positions having two different X-Y co-ordinates: one of the spots (H032.0) was identified as phosphorylated Era, indicating that a substantial portion of Era in the cell is indeed phosphorylated. Therefore, Era autophosphorylation is likely to play an important physiological role in the cell. The sequence encoding the C-terminus previously published had a missing C between A900 and GgO1. As a resuit of the frameshift, Era consists of 301 residues, 15 fewer than originaiiy reported.     

Characterization of mutations affecting the Escherichia coli essential GTPase era that suppress two temperature-sensitive dnaG alleles.

Two suppressor mutations of the temperature-sensitive DNA primase mutant dnaG2903 have been characterized. The gene responsible for suppression, era, encodes an essential GTPase of Escherichia coli. One mutation, rnc-15, is an insertion of an IS1 element within the leader region of the rnc operon and causes a polar defect on the downstream genes of the operon. A previously described polar mutation, rnc-40, was also able to suppress dnaG2903. The other mutation, era-1, causes a single amino acid substitution (P17R) in the G1 region of the GTP-binding domain of Era. Analysis of the GTPase activity of the Era-1 mutant protein showed a four- to five-fold decrease in the ability to convert GTP to GDP. Thus, lowered expression of wild-type Era caused by the polar mutations and reduced GTPase activity caused by the era-1 mutation suppresses dnaG2903 as well as a second dnaG allele, parB. Phenotypic analysis of the era-1 mutant at 25 degrees C showed that 10% of the cells contain four segregated nucleoids, indicative of a delay in cell division. Possible mechanisms of suppression of dnaG and roles for Era are discussed.     

Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli

Phenotypic analysis of a temperature-sensitive era mutant strain indicates that Escherichia coli cells depleted of Era undergo many physiological changes. At 43 degrees C, a completely non-permissive temperature, growth is arrested because of loss of the gene and depletion of the Era protein. Depletion of Era at 43 degrees C results in depressed synthesis of heat-shock proteins DnaK, GroEL/ES, D33.4 and C62.5, lack of thermal induction of ppGpp pool levels, and increased capacity for carbon source metabolism through the citric acid cycle. Thus, in addition to inhibition of cell growth and viability, loss of Era function results in pleiotropic changes including abnormal adaptation to thermal stress.     

Cold-sensitive growth and decreased GTP-hydrolytic activity from substitution of Pro17 for Val in Era, an essential Escherichia coli GTPase

A substitution mutation of Pro17 by Val (P17V) was constructed in the guanine nucleotide binding domain of Era, an essential protein in Escherichia coli. The mutation is analogous to the oncogenic activating allele at position 12 in the GTP-binding domain of p21ras. The phenotype of this mutant was analysed in a strain which exclusively expressed the mutant protein (Era-V17) in null allele chromosomal background (era1: :kan). The strain was found to be cold-sensitive for growth. Mutant Era-V17 purified from the strain was cold-sensitive for GTP-hydrolytic activity, suggesting that the GTPase activity of Era is required for cell growth since the P17V mutation resulted in both cold-sensitive growth of cells and cold-labile GTPase activity of the purified protein.     

Isolation and characterization of temperature-sensitive and thermostable mutants of the human receptor-like protein tyrosine phosphatase LAR.

Human LAR is a transmembrane receptor-like protein whose cytoplasmic region contains two tandemly duplicated domains homologous to protein tyrosine phosphatases (PTPases). Whereas the membrane-proximal domain I has enzymatic activity, the membrane-distal domain II has no apparent catalytic activity but seems to have a regulatory function. In order to study structure-function relationships of the LAR PTPase, LAR domain I was expressed in Escherichia coli, and mutants that have reduced catalytic activity or reduced thermostability were isolated and characterized. We isolated 18 unique hydroxylamine-induced missense mutations in the LAR domain I segment, of which three were temperature-sensitive. Five additional temperature-sensitive mutations were isolated using N-methyl-N'-nitro-N-nitrosoguanidine. All eight temperature-sensitive mutations are confined within a short segment of the LAR domain I sequence between amino acid positions 1329 and 1407. To examine whether this region is particularly prone to temperature-sensitive mutations, tyrosine at amino acid position 1379 was changed to a phenylalanine by oligonucleotide-directed mutagenesis. This mutant, Y1379-F, was indeed temperature-sensitive. We also isolated a revertant of a temperature-sensitive mutant. The revertant contained a second-site mutation (C1446-Y) that suppresses several temperature-sensitive mutations and also enhances the folding of LAR protein produced in E. coli.     

Unique temperature-sensitive defect in vaccinia virus morphogenesis maps to a single nucleotide substitution in the A30L gene

Marker rescue experiments demonstrated that the genetic lesion of a previously isolated vaccinia virus temperature-sensitive mutant which forms multilayered envelope structures with lucent interiors and foci of viroplasm with dense centers mapped to the A30L open reading frame. A single base change, resulting in a nonconservative Ser-to-Phe substitution at residue 17, was associated with degradation of the A30L protein at elevated temperatures.     

A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.     

Recombinant-derived interleukin-1 alpha stabilized against specific deamidation

Recombinant-derived human interleukin-1 alpha (IL-1 alpha), purified from Escherichia coli, was resolved by isoelectric focusing on polyacrylamide gels into two species of isoelectric points (pI) 5.45 and 5.20, which constituted approximately 75% and approximately 25% of the total IL-1 alpha protein respectively. The pI 5.45 and pI 5.20 species were separated by chromatofocusing and subjected to N-terminal sequence analysis. The pI 5.45 species contained the expected Asn residue at position 36 of the mature protein sequence whereas the pI 5.20 species contained an Asp residue at the same position. A mutant protein in which Asn-36 was substituted for a Ser residue was isolated from E. coli and shown to be homogeneous on isoelectric focusing analysis with a pI = 5.45. 1H-n.m.r. and circular dichroism analyses of wild-type and the mutant IL-1 alpha indicated a similar conformation which was also indicated by the identical receptor binding affinities of IL-1 alpha with Asn, Asp or Ser in position 36. The mutant protein was stabilized against specific base-catalysed and temperature-induced deamidation, and may be more suitable than the wild-type position for physical and structural studies.     

Temperature-sensitive cloning vector for Corynebacterium glutamicum

We constructed a temperature-sensitive form of the Corynebacterium glutamicum ATCC13869 cryptic plasmid, pBL1. The C. glutamicum/Escherichia coli shuttle vector pSFK6, which is composed of pBL1 and the E. coli cloning vector pK1, was mutagenized in vitro by treatment with hydroxylamine, and introduced into C. glutamicum cells. A mutant plasmid, which was stably maintained at 25 degrees C but not at 34 degrees C, was isolated from the cells. Sequencing the plasmid, which was named p48K, revealed four substitutions in the Rep protein coding region. Moreover, site-directed single-nucleotide substitutions showed that a G to A transition at position 2,920, which resulted in a Pro-47 to Ser substitution in the Rep protein, was responsible for its temperature-sensitive replication. Pro-47 is conserved among the Rep proteins of the pIJ101/pJV1 family of plasmids. This temperature-sensitive cloning vector will be useful for disrupting genes in this industrially important bacterium.     

Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli.

Temperature-resistant pseudorevertants of the temperature-sensitive Escherichia coli mutant KNS19, harboring a mutation in rplX, the gene for ribosomal protein L24, were isolated, cloned, and sequenced. The codon GAC for the amino acid Asp in the temperature-sensitive mutant corresponding to position 84 in the protein chain mutated either back to the wild type (Gly) or to codons for the amino acids Tyr and Glu. Furthermore, rplX genes from two other mutants with an altered protein L24 were cloned and sequenced. The mutations were localized at position 56 (Gly to Asp) and at position 62 (Glu to Lys) in the rplX gene. The latter two mutants lacked a conditional lethal phenotype. The results suggest that the amino acid Gly at positions 56 and 84 in the protein might be involved in loop formations.     

An initiator protein for plasmid R6K DNA replication. Mutations affecting the copy-number control

Two kinds of mutations affecting the copy-number control of plasmid R6K were isolated and identified in an initiator pi protein by DNA sequencing. Firstly, a temperature-sensitive replication mutation, ts22, with decreased copy number results in a substitution of threonine to isoleucine at position 138 of the 305-amino-acid pi protein. Secondly, a high-copy-number (cop21) mutant was isolated from this ts mutant and was identified by an alteration of alanine to serine at position 162. This cop21 mutation suppressed the Ts character and was recessive to the wild-type allele in the copy control.     

A temperature-sensitive mutant in the gene rplX for ribosomal protein L24 and its suppression by spontaneous mutations in a 23S rRNA gene of Escherichia coli.

A temperature-sensitive mutant with an altered ribosomal protein L24 was analysed. Revertant analysis showed that the temperature-sensitive growth was correlated with the altered protein. A DNA segment containing the mutant rplX gene was cloned and sequenced. The GGC codon for glycine at the amino acid position 84 of the protein was found to be altered to a GAC codon for aspartic acid. By transforming the rplX mutant with a plasmid carrying the rrnB operon and by selecting for temperature-resistant transformants we obtained two spontaneous suppressor mutants in the gene for 23S rRNA. DNA sequence analysis of the region corresponding to the 5' end of the 23S rRNA showed a C to T alteration at position 33 in both mutants and an additional A to G alteration at position 466 in one of them. The results suggest intimate interaction of protein L24 and the 5' end of 23S rRNA in vivo and support a secondary structure model of the 23S rRNA which brings these mutational points into a close contact.     

Two mutant alleles of mukB, a gene essential for chromosome partition in Escherichia coli

Abstract The MukB protein is essential for chromosome partitioning in Escherichia coli and consists of 1484 amino acid residues (170 kDa). We have determined the base changes at the mutated sites of the mukB106 mutant and a newly isolated mutant, mukB33 . These mutant mukB genes were each found to carry a single base-pair transition which leads to an amino acid substitution; a serine residue at position 33 was changed to phenylalanine in the case of mukB106 , and an aspartic acid residue at position 1201 was changed to asparagine in the case of mukB33 .     

Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome

Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.     

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.     

Molecular characterization of the Escherichia coli htrD gene: cloning, sequence, regulation, and involvement with cytochrome d oxidase.

The Escherichia coli htrD gene was originally isolated during a search for new genes required for growth at high temperature. Insertional inactivation of htrD leads to a pleiotropic phenotype characterized by temperature-sensitive growth in rich medium, H2O2 sensitivity, and sensitivity to cysteine. The htrD gene was cloned and sequenced, and an htrD::mini-Tn10 insertion mutation was mapped within this gene. The htrD gene was shown to encode a protein of approximately 17.5 kDa. Expression of the htrD gene was examined by using an phi (htrD-lacZ) operon fusion. It was found that htrD is not temperature regulated and therefore is not a heat shock gene. Further study revealed that htrD expression is increased under aerobic growth conditions. Conversely, under anaerobic growth conditions, htrD expression is decreased. In addition, a mutation within the nearby cydD gene was found to drastically reduce htrD expression under all conditions tested. These results indicate that htrD is somehow involved in aerobic respiration and that the cydD gene product is necessary for htrD gene expression. In agreement with this conclusion, htrD mutant bacteria are unable to oxidize the cytochrome d-specific electron donor N,N,N',N'-tetramethyl-p-phenylenediamine.     

Enhanced Tn10 and mini-Tn10 precise excision in DNA replication mutants of Escherichia coli K12

The precise excision of transposon Tn10 and a mini-Tn10 derivative, inserted in the gal or lac operons, was studied in dnaB252 and dnaE486 temperature-sensitive mutants of Escherichia coli. dnaB codes for a DNA replication helicase and dnaE for the alpha subunit of DNA polymerase III. Mutations in these genes were found to enhance, at the permissive temperature, the precise excision of both genetic elements. The increase factor was much more pronounced for the dnaB252 mutant with the transposons inserted in gal. The stimulated excision was only partially affected by a recA null mutation but was significantly reduced by introduction of recF null or ruvA mutations. A model involving template switching of the polymerase between the direct repeats flanking the transposons, on the same strand or between sister strands, could account for the observed results.     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.