Identification, characterization, and mapping of the Escherichia coli htrA gene, whose product is essential for bacterial growth only at elevated temperatures.

We identified and cloned an Escherichia coli gene called htrA (high temperature requirement). The htrA gene was originally discovered because mini-Tn10 transposon insertions in it allowed E. coli growth at 30 degrees C but prevented growth at elevated temperatures (above 42 degrees C). The htrA insertion mutants underwent a block in macromolecular synthesis and eventually lysed at the nonpermissive temperature. The htrA gene was located at approximately 3.7 min (between the fhuA and dapD loci) on the genetic map of E. coli and between 180 and 187.5 kilobases on the physical map. It coded for an unstable, 51-kilodalton protein which was processed by removal of an amino-terminal fragment, resulting in a stable, 48-kilodalton protein.     

Isolation and characterization of the Escherichia coli htrB gene, whose product is essential for bacterial viability above 33 degrees C in rich media.

We have identified and studied the htrB gene of Escherichia coli. Insertional inactivation of the htrB gene leads to bacterial death at temperatures above 33 degrees C. The mutant bacterial phenotype at nonpermissive temperatures includes an arrest of cell division followed by the formation of bulges or filaments. The htrB+ gene has been cloned by complementation and shown to reside at 23.4 min on the E. coli genetic map, the relative order of the neighboring loci being mboA-htrB-pyrC. The htrB gene is transcribed in a counterclockwise fashion, relative to the E. coli genetic map, and its product has been identified as a membrane-associated protein of 35,000 Da. Growth experiments in minimal media indicate that the HtrB function becomes dispensable at low growth rates.     

Low temperature-induced systems failure in Escherichia coli: insights from rescue by cold-adapted chaperones

The growth of Escherichia coli cells is impaired at temperatures below 21 degrees C and stops at 7.5 degrees C; however, growth of a transgenic strain producing the cold-adapted chaperones Cpn60 and Cpn10 from the psychrophilic bacterium Oleispira antarctica is good at low temperatures. The E. coli cpn(+) transgene offers a novel opportunity for examining the essential protein for cell viability at low temperatures. By screening a large-scale protein map (proteome) of cells of K-12 and its Cpn(+) transgene incubated at 4 degrees C, we identified 22 housekeeping proteins involved in systems failure of E. coli when confronted with low temperature. Through co-immunoprecipitation of Cpn60, Northern blot, and in vitro refolding, we systematically identified that protein-chaperone interactions are key determinants of their protein functions at low temperatures. Furthermore, chromosomal gene deletion experiments suggest that the mechanism of cold-induced systems failure in E. coli is cold-induced inactivation of the GroELS chaperonins and the resulting failure to refold cold-inactivated Dps, ClpB, DnaK and RpsB proteins. These findings: (1) indicate the potential importance of chaperones in cold sensitivity, cold adaptation and cold tolerance in cellular systems, and (2) suggest the identity of a few key cold-sensitive chaperone-interacting proteins that get inactivated and ultimately cause systems failure in E. coli cells at low temperatures.     

Analysis of an Escherichia coli dnaB temperature-sensitive insertion mutation and its cold-sensitive extragenic suppressor.

An Escherichia coli mutant, ts121, was isolated following random insertional mutagenesis using phage lambda Mu transposition. The mutant phenotype includes inability to form colonies at temperatures above 38 degrees C and inability to propagate phage lambda at all temperatures. A lambda i434 cI- (ts121)+ transducing phage was isolated on the basis of its ability to form plaques on ts121 mutant bacteria. Using this transducing phage, it was shown through complementation and protein analyses, that the ts121 mutation is located in the dnaB gene. The exact insertion event was identified by polymerase chain reaction amplification of the DNA sequences containing the insertion junction. The mutational insertion event in ts121 was mapped precisely between base pairs 1514 and 1515 of the dnaB gene. This result predicts that the mutant dnaB protein has lost its six terminal amino acids. The reading frame shifts into Mu-specific DNA sequences resulting in an additional 20 amino acid residues. The E. coli wild type dnaB protein participates in host replication and interacts with lambda P protein to initiate phage lambda DNA replication. Our results demonstrate that the extreme carboxyl end of the dnaB protein is required for productive interaction with the lambda P replication protein at all temperatures, and is important for dnaB function at temperatures above 38 degrees C. Cold-sensitive extragenic suppressors of the ts121 mutation were isolated on the basis of their ability to restore colony formation at 42 degrees C. One of these extragenic suppressors was mapped at 54 min on the E. coli genetic map and localized to the suhB gene, whose product may affect the expression of a number of genes at the translational level.     

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.     

Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB.

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.     

A single base change in the acceptor stem of tRNA(3Leu) confers resistance upon Escherichia coli to the calmodulin inhibitor, 48/80

We have isolated several classes of spontaneous mutants resistant to the calmodulin inhibitor 48/80 which inhibits cell division in Escherichia coli K12. Several mutants were also temperature sensitive for growth and this property was exploited to clone a DNA fragment from an E. coli gene library restoring growth at 42 degrees C and drug sensitivity at 30 degrees C in one such mutant. Physical and genetic mapping confirmed that both the mutation and the cloned DNA were located at 15.5 min on the E. coli chromosome at a locus designated feeB. By subcloning, complementation analysis and sequencing, the feeB locus was identified as identical to the tRNA(CUALEU) gene. When the mutant locus was isolated and sequenced, the mutation was confirmed as a single base change, C to A, at position 77 in the acceptor stem of this rare Leu tRNA. In other studies we obtained evidence that this mutant tRNA, recognizing the rare Leu codon, CUA, was defective in translation at both permissive and non-permissive temperatures. The feeB1 mutant is defective in division and shows a reduced growth rate at non-permissive temperature. We discuss the possibility that the mutant tRNA(3Leu) is limiting for the synthesis of a polypeptide(s), requiring several CUA codons for translation which in turn regulates in some way the level or activity of the drug target, a putative cell cycle protein.     

Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium

The gene encoding alkaline phosphatase from the psychrotrophic bacterium Shewanella sp. SIB1 was cloned, sequenced, and overexpressed in Escherichia coli. The recombinant protein was purified and its enzymatic properties were compared with those of E. coli alkaline phosphatase (APase), which shows an amino acid sequence identity of 37%. The optimum temperature of SIB1 APase was 50 degrees C, lower than that of E. coli APase by 30 degrees C. The specific activity of SIB1 APase at 50 degrees C was 3.1 fold higher than that of E. coli APase at 80 degrees C. SIB1 APase lost activity with a half-life of 3.9 min at 70 degrees C, whereas E. coli APase lost activity with a half-life of >6 h even at 80 degrees C. Thus SIB1 APase is well adapted to low temperatures. Comparison of the amino acid sequences of SIB1 and E. coli APases suggests that decreases in electrostatic interactions and number of disulfide bonds are responsible for the cold-adaptation of SIB1 APase.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).     

Isolation and characterization of a temperature-sensitive conditional mutant of Escherichia coli altered for the control of phosphate-regulated proteins

We have identified a conditional mutation which confers a ple?otropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C. This mutation was located at minute 89.5 on the E. coli chromosome in a gene we have called cpr for conditional phosphate-regulated.     

Cloning and characterization of Bacillus subtilis homologs of Escherichia coli cell division genes ftsZ and ftsA.

The Bacillus subtilis homolog of the Escherichia coli ftsZ gene was isolated by screening a B. subtilis genomic library with anti-E. coli FtsZ antiserum. DNA sequence analysis of a 4-kilobase region revealed three open reading frames. One of these coded for a protein that was about 50% homologous to the E. coli FtsZ protein. The open reading frame just upstream of ftsZ coded for a protein that was 34% homologous to the E. coli FtsA protein. The open reading frames flanking these two B. subtilis genes showed no relationship to those found in E. coli. Expression of the B. subtilis ftsZ and ftsA genes in E. coli was lethal, since neither of these genes could be cloned on plasmid vectors unless promoter sequences were first removed. Cloning the B. subtilis ftsZ gene under the control of the lac promoter resulted in an IPTGs phenotype that could be suppressed by overproduction of E. coli FtsZ. These genes mapped at 135 degrees on the B. subtilis genetic map near previously identified cell division mutations.     

lon gene product of Escherichia coli is a heat-shock protein

The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0 in E. coli K-12 strains. Peptide map patterns of Lon protease and H94.0 were identical. A mutant form of the protease had altered mobility during gel electrophoresis. An E. coli B/r strain that is known to be defective in Lon function contained no detectable H94.0 protein under normal growth conditions. Upon a shift to 42 degrees C, however, the Lon protease was induced to high levels in K-12 strains and a small amount of protein became detectable at the H94.0 location in strain B/r. Heat induction of Lon protease was dependent on the normal allele of the regulatory gene, htpR, establishing lon as a member of the high-temperature-production regulon of E. coli.     

CspI, the ninth member of the CspA family of Escherichia coli, is induced upon cold shock

Escherichia coli contains the CspA family, consisting of nine proteins (CspA to CspI), in which CspA, CspB, and CspG have been shown to be cold shock inducible and CspD has been shown to be stationary-phase inducible. The cspI gene is located at 35.2 min on the E. coli chromosome map, and CspI shows 70, 70, and 79% identity to CspA, CspB, and CspG, respectively. Analyses of cspI-lacZ fusion constructs and the cspI mRNA revealed that cspI is cold shock inducible. The 5'-untranslated region of the cspI mRNA consists of 145 bases and causes a negative effect on cspI expression at 37 degrees C. The cspI mRNA was very unstable at 37 degrees C but was stabilized upon cold shock. Analyses of the CspI protein on two-dimensional gel electrophoresis revealed that CspI production is maximal at or below 15 degrees C. Taking these results together, E. coli possesses a total of four cold shock-inducible proteins in the CspA family. Interestingly, the optimal temperature ranges for their induction are different: CspA induction occurs over the broadest temperature range (30 to 10 degrees C), CspI induction occurs over the narrowest and lowest temperature range (15 to 10 degrees C), and CspB and CspG occurs at temperatures between the above extremes (20 to 10 degrees C).     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

Thermostable repair enzyme for oxidative DNA damage from extremely thermophilic bacterium, Thermus thermophilus HB8.

The mutM (fpg) gene, which encodes a DNA glycosylase that excises an oxidatively damaged form of guanine, was cloned from an extremely thermophilic bacterium, Thermus thermophilus HB8. Its nucleotide sequence encoded a 266 amino acid protein with a molecular mass of approximately 30 kDa. Its predicted amino acid sequence showed 42% identity with the Escherichia coli protein. The amino acid residues Cys, Asn, Gln and Met, known to be chemically unstable at high temperatures, were decreased in number in T.thermophilus MutM protein compared to those of the E.coli one, whereas the number of Pro residues, considered to increase protein stability, was increased. The T.thermophilus mutM gene complemented the mutability of the E.coli mutM mutY double mutant, suggesting that T. thermophilus MutM protein was active in E.coli. The T.thermophilus MutM protein was overproduced in E.coli and then purified to homogeneity. Size-exclusion chromatography indicated that T. thermophilus MutM protein exists as a more compact monomer than the E.coli MutM protein in solution. Circular dichroism measurements indicated that the alpha-helical content of the protein was approximately 30%. Thermus thermophilus MutM protein was stable up to 75 degrees C at neutral pH, and between pH 5 and 11 and in the presence of up to 4 M urea at 25 degrees C. Denaturation analysis of T.thermophilus MutM protein in the presence of urea suggested that the protein had at least two domains, with estimated stabilities of 8.6 and 16.2 kcal/mol-1, respectively. Thermus thermophilus MutM protein showed 8-oxoguanine DNA glycosylase activity in vitro at both low and high temperatures.