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Journal of Bacteriology, December 2006, p. 8317-8320, Vol. 188, No. 23
0021-9193/06/$08.00+0     doi:10.1128/JB.00977-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

The Escherichia coli rpoS-Dependent htrC Gene Is Not Involved in the Heat Shock Response

Zubin Thacker,¹ Elise Darmon,¹ France Keppel,² and Millicent Masters^1*

University of Edinburgh, School of Biology, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland,¹ Département de microbiologie et médecine moléculaire, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneve 4, Suisse²

Received 5 July 2006/ Accepted 7 September 2006

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We found that a new mutant with a deletion/replacement of the Escherichia coli K-12 htrC gene, a gene previously reported to be required for growth at elevated temperatures, is not temperature sensitive. Furthermore, the original mutants, kindly provided by the original authors, although temperature sensitive, do not have mutations in the open reading frame designated htrC. We found that htrC requires RpoS for enhanced expression in the early stationary phase and is expressed at very low levels until then. The growth of our htrC mutant slowed during the early stationary phase, and the mutant was replaced by its parent in mixed cultures. Since we cannot assign a function or distinctive phenotype to htrC, we suggest that this open reading frame should be given a positional designation, yjaZ, until a specific function is identified.

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htrC was first described as an Escherichia coli heat shock gene by Raina and Georgopoulos in 1990 (10). These authors reported that transposon insertions in, or deletion of, htrC caused temperature sensitivity at 42°C accompanied by extensive filamentation and lysis. Expression of htrC was reported to be controlled by the "heat shock" sigma factor, ³²; htrC mutants also overproduced heat shock proteins, such as DnaK, GroEL, GrpE, and HtpG, at 30°C. Since the initial report, substantive further information about htrC has been published only in other papers whose authors included Raina and Georgopoulos (2, 7).

HtrC came to our attention as one of the small number of proteins apparently limited to E. coli and its closest relatives. Most of the other E. coli-specific proteins have unknown functions; mutants with mutations in these proteins grow normally on LB broth at 37°C and also grow at higher temperatures (11). Since htrC stood out in this group as a reportedly essential gene with an important biological role, it appeared to warrant further examination.

htrC is not involved in the heat shock response. The MG1655 htrC gene is located between rpoC and thiCEFSGH. To verify the previously published phenotype, a new mutant of htrC (htrC), in which most of the htrC coding region was replaced with a removable cassette, FLK2, containing a lacZ reporter, a selectable aph (Kan^r) marker, and a lac promoter to facilitate downstream transcription, was constructed using the method described by Merlin and coworkers (5). PCR analysis, using primers both within and outside the deleted region, was used to verify that the htrC mutant indeed lacks 84% of the htrC coding region and that the chromosome does not contain an intact copy of htrC in addition to the mutated allele. Surprisingly, the phenotypic properties of the mutant were not the same as the properties reported previously. The viability on LB agar at 43 or 45°C was equal to that of the parent strain. The growth rates of MG1655 and MG1655htrC in LB broth at 30, 37, and 43°C were also found to be similar (Fig. 1A). Furthermore, in contrast to previous reports, both in MG1655 (Fig. 2) and in W3110 and MC4100 htrC mutants, induction at 42°C of the representative heat shock proteins GroEL and GroES was unaffected. As these results did not agree with the previously described results, we decided to reexamine the original mutants. Three strains, the parent strain CA8000 and two kanamycin-resistant putative transposon mutants of htrC identified as 206 and 280 (10), were received from S. Raina. The growth rates of CA8000, 206, and 280 were determined at 30 and 43°C to verify the previously described phenotypes; 206 and 280 were temperature sensitive as reported previously.

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FIG. 1. Growth of MG1655 and MG1655htrC in LB broth at 30, 37, and 43°C and expression of htrC in the mutant strain. (A) Cultures of MG1655 and MG1655htrC were grown exponentially at 30°C for three generations and then diluted, and growth was monitored at 30, 37, and 43°C. Symbols: , 30°C; •, 37°C; , 43°C. Solid symbols, MG1655; open symbols, MG1655htrC. (Note that there are as many open symbols as solid symbols, although because the two often coincide, they are not separately visible; the 37°C data are from a separate experiment.) (B) Transcription from htrC in MG1655htrC, measured by determining the ß-galactosidase activity in Miller units (6) and plotted against culture growth in units of optical density at 600 nm (OD 600nm). The symbols are as described above for panel A, except that 37°C data are indicated by triangles. (C) Total ß-galactosidase activity per aliquot of culture rather than per unit of optical density at 600 nm. The symbols are as described above for panel B.

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FIG. 2. Induction of heat shock proteins in htrC and htrC+ MG1655 strains. Bacteria were grown at 30°C to an optical density at 600 nm of 0.15 and then incubated at 30 or 42°C for 15 min. Proteins were extracted and separated on a 13% sodium dodecyl sulfate-polyacrylamide gel as described by Laemmli (3), stained with Coomassie brilliant blue, and photographed. Lane 1, 30°C, htrC+; lane 2, 30°C, htrC; lane 3, 42°C, htrC+; lane 4, 42°C, htrC.

Linkage between the htrC gene and the temperature sensitivity of mutant strains 206 and 280 was tested by examining the linkage between tetracycline resistance on a Tn10 transposon in argE (CAG12185) (9) (supplied by M. Berlyn) and the kanamycin resistance gene putatively in htrC in strains 206 and 280. argE is located approximately 1 min from htrC and would be expected to cotransduce with htrC⁺ at a frequency of 20% to yield Tet^r Kan^s progeny. Strains 206 and 280 were transduced with a lysate prepared from CAG12185, and 100 tetracycline-resistant progeny from each transduction were purified on LB medium plates containing tetracycline and screened for kanamycin resistance. All Tet^r transductants remained Kan^r, indicating that the transposon was unlikely to be within 2 min of argE.

To determine whether the aph gene in the 206 and 280 strains is linked to the locus causing temperature sensitivity, the temperature-sensitive mutants were transduced to temperature resistance using P1 lysates prepared from MG1655. All 100 temperature-resistant progeny screened for each parent were still Kan^r, showing that the two phenotypes, temperature sensitivity and kanamycin resistance, are not closely linked by P1 transduction.

The Tn5 aph gene was shown to be present in 206 and 280 by PCR amplification of a 950-bp Tn5 fragment including the aph gene. Since the transductional studies described above did not eliminate the possibility that 206 and 280 contain more than one Tn5, only one of which is in htrC, lysates were prepared for 206 and 280 and used to transduce CA8000. At least 120 Kan^r progeny were purified from each cross and tested to determine their temperature sensitivity. All progeny were temperature resistant, indicating that temperature sensitivity is unlikely to be linked to any copy of Tn5 that 206 or 280 harbors. For further confirmation that the htrC gene in 206 or 280 was not interrupted by a Tn5 insertion, as had been reported previously (10), the htrC genes in these strains were sequenced. The sequences obtained indicated that the sequence of htrC is neither interrupted nor altered. We therefore concluded that temperature-sensitive mutants 206 and 280 are not temperature-sensitive because of mutation of the htrC gene. Although these strains do carry a Tn5-based kanamycin resistance gene, it is not linked to temperature sensitivity or to htrC.

Seeking a phenotype for htrC. The growth of MG1655 and the growth of MG1655htrC were compared under a variety of conditions. The strains were streaked and incubated at 37°C on LB medium plates containing NaCl (800 or 1,200 mM) or metals (Zn or Ni at a concentration of 2 or 3 mM, Co at a concentration of 1 or 1.5 mM, and Cu at a concentration of 5 or 6 mM) or at different pH values (pH 5.6, 5.8, 9, or 9.2). No differences in viability or the rate of colony growth were observed. Colony formation on minimal glucose medium at 30 or 37°C was also normal. To be certain that the host that we used did not contain a suppressor of the htrC phenotype, the htrC deletion/substitution was P1 transduced, selecting for the Kan^r marker in the cassette, to CA8000, the strain originally used to isolate htrC mutants (9). CA8000 htrC mutants were also found to grow normally in LB broth at 30 and 43°C. In a further attempt to demonstrate a mutant phenotype, MG1655 and its isogenic htrC mutant were grown together in a mixed culture to see if the mutant would be replaced by the parent. Equal volumes of overnight stationary-phase cultures of the strains were mixed and diluted 5 x 10⁴-fold into fresh LB medium for further overnight growth. The next day samples of the cultures were plated so that the proportions of parent and mutant (Kan^r) colonies could be determined. Dilution, growth, and plating were repeated a total of six times. In each of four replicates the parental strain gradually replaced the htrC mutant, whose level declined about 25% per passage (Fig. 3A). Since the viability of neither the wild type nor the htrC strain decreased in stationary cultures left for several weeks at room temperature, the mutant must grow more slowly during some part of its life cycle. We therefore looked at growth of the strains more carefully and found that the growth of the htrC mutant slowed relative to the growth of the parent during the early stationary phase. Figure 3B shows that growth of the htrC mutant slowed about one generation earlier than growth of the parent slowed. The growth curves, which were superimposable previously, were parallel after the growth of both strains slowed. If the slower strain is one generation behind the faster strain, so that these strains comprise 66% and 33% of the population, respectively, when growth ceases, this is equivalent to a loss of about one-third of the slower strain per passage, which is broadly in agreement with what was observed. To be certain that the relative disadvantage of the htrC mutant was not caused by the presence of the cassette that we substituted for the htrC coding sequence, the temperature-sensitive plasmid pCP20 (1), containing the FLP recombinase, was introduced into htrC; Kan^s progeny, from which FLK2 was presumed to be deleted, were identified, and pCP20 was cured by growth at a high temperature. The growth in LB broth of this strain, htrC FLK2 (which retained only a 93-bp in-frame scar), was compared with the growth of its parent and MG1655, and its growth was also found to slow early in the stationary phase (Fig. 3B). We therefore deduced that the slowing of growth is probably attributable to lack of HtrC rather than to the presence of FLK2.

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FIG. 3. Coculture and growth of MG1655 and its htrC derivative. (A) LB broth cultures of the two strains were grown, mixed, diluted, and analyzed as described in the text. Unique symbols are used for the four replicates. (B) Growth at 37°C of two separate cultures of MG1655 (MG1655-1 and MG1655-2) and of MG1655 in which htrC was replaced by the FLK2 cassette or by the 93-bp scar left by its removal. Growth curves were normalized to zero time at an optical density at 600 nm [OD (600nm)] of 1.0.

Expression of htrC was measured by determining the level of ß-galactosidase produced from the lacZ reporter cassette under the control of the native htrC promoter as described by Miller (6). Overnight cultures of MG1655 and its htrC mutant were diluted, grown for three generations at 30°C to an optical density of 0.2 to 0.3, and again diluted into LB broth. Growth at 30, 37, and 43°C (Fig. 1A) and gene expression were monitored (Fig. 1B and C). The level of expression of htrC during exponential growth was very low at all temperatures, and there was only a slightly higher level of expression at 43°C. In another experiment (data not shown) expression was also measured after a shift to 50°C, since it was reported previously that the htrC message was strongly expressed at this temperature; the level of expression remained unchanged. This is consistent with the recently reported work of Nonaka et al. (9), who failed to find evidence of htrC induction in a genome-wide survey of genes under ³² control. We did observe, however, that as the cultures approached the stationary phase at 37 and 43°C (growth of the 30°C culture was not monitored to the stationary phase), htrC expression increased rather abruptly, increasing approximately fivefold by the time that growth ceased.

This was true whether the enzyme level was measured by determining the differential rate of synthesis (Fig. 1B) or by determining the total amount of enzyme synthesized (Fig. 1C), showing that the synthesis of the enzyme actually increased rather than merely slowed less quickly than the synthesis of other proteins. The high levels of transcription reached in stationary phase could also be inferred from the high specific activities observed in the diluted overnight cultures at the start of growth (Fig. 1B). The increased expression of htrC in stationary phase suggests that htrC might be under the control of RpoS, a sigma factor regulating genes expressed in the stationary phase and important in some stress responses. This was tested by using P1 to transduce the rpoS359::Tn10 marker from RH90 (4) to MG1655htrC. Figure 4 shows that stationary-phase expression of htrC is indeed dependent on an intact rpoS gene. Together with the slow growth of the mutant in the stationary phase, this suggests that HtrC may have a role in maintaining the growth rate when cell density is high.

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FIG. 4. Growth of and htrC expression in MG1655htrC and its rpoS derivative at 37°C. OD 600nm, optical density at 600 nm.

Distribution of htrC among bacteria. Strikingly, the 540-bp htrC open reading frame, which encodes a very basic protein, is rich in class 3 codons, which is typical for a gene acquired recently by horizontal transfer (http://genolist.pasteur.fr/Colibri/). A BLAST search revealed unequivocal htrC homology in only a small number of enterobacterial species. For the eight genera of free-living Enterobacteriacae with one or more fully sequenced species, only two of three E. coli subspecies and two of four Shigella species have an htrC homolog. The E. coli O157:H7 isolates have a significant deletion near the C terminus of the gene, although the Shigella homologs are intact. Although a protein with possibly significant amino acid identity or similarity was found in several other -proteobacteria, the next most similar protein found in the BLAST search was the protein of Verminephobacter, which, interestingly, is a ß-proteobacterial symbiont of a worm rather than a -proteobacterium. We believe that together, this information suggests that there was recent acquisition followed by loss in the enterobacterial line rather than more ancient acquisition.

Although it seems clear that an intact rpoS gene is required for significant levels of htrC expression, an important role in stationary-phase metabolism seems improbable for a gene so poorly distributed and likely to have been recently acquired. We cannot exclude the possibility that the transcription observed in stationary phase is transcription from a promoter next to which htrC has been inserted by chance or that the small growth disadvantage that the mutant displays in stationary phase is a consequence of a positional effect on another gene rather than of a lack of HtrC protein.

In summary, we found that deletion of htrC does not result in temperature sensitivity and that htrC is expressed at a very low rate during exponential growth, even at high temperatures, consistent with the hypothesis that the heat shock sigma factor plays no role in its expression. As htrC does not appear to have a role in the heat shock response of E. coli, we believe that htrC is not a suitable mnemonic for this gene. Since the limited number of tests that we have done to determine the function of htrC did not identify any pronounced mutant phenotype, we suggest that for the time being htrC should be reclassified as a gene with an unknown function and that it should be given the positional designation yjaZ. Clearly, further experiments are required to identify a possible function of htrC in E. coli K-12.

 
This work was supported by the Darwin Trust (Z.T.), by Swiss National Foundation grant FN-31-65403 to Costa Georgopoulos (F.K.), and by an MRC grant to David Leach (E.D.).

We thank Costa Georgopoulos for encouraging us to pursue this line of investigation and for critically reading the manuscript.

 
* Corresponding author. Mailing address: University of Edinburgh, School of Biology, Darwin Building, King's Building, Mayfield Road, Edinburgh EH9 3JR, Scotland. Phone: 44 (0)131 650 5355. Fax: 44 (0)131 650 8650. E-mail: M.Masters{at}ed.ac.uk.

Published ahead of print on 15 September 2006.

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Journal of Bacteriology, December 2006, p. 8317-8320, Vol. 188, No. 23
0021-9193/06/$08.00+0     doi:10.1128/JB.00977-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Thacker, Z. Articles by Masters, M. Search for Related Content PubMed PubMed Citation Articles by Thacker, Z. Articles by Masters, M.