The Pseudomonas aeruginosa hscA gene encodes Hsc66, a DnaK homologue
Jesús Campos-García1,
Leandro G. Ordóñez1 and
Gloria Soberón-Chávez1
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo Postal 510-3, Cuernavaca, Morelos, Mexico1
Author for correspondence: Gloria Soberón-Chávez. Tel:+52 73 291634. Fax: +52 73 172388. e-mail: gloria{at}ibt.unam.mx
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ABSTRACT
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Under heat-stress conditions bacteria induce, among other heat-shock proteins, the Hsp70 molecular chaperone (DnaK), which is involved in protein stabilization. It has been shown in Escherichia coli that an Hsp70 homologue called Hsc66, which is widespread in bacteria, functions as a chaperone in vitro. This paper reports the isolation of a Pseudomonas aeruginosa W51D mutant (W51M22) by insertion of the mini-Tn5-Hg transposon, which was unable to grow on ethanol and other short-chain alcohols as sole source of carbon. The transposon insertion in this mutant was shown to be located in the hscA gene encoding Hsc66. The inability of mutant W51M22 to use ethanol was complemented by the E. coli hscBAfdx operon. The authors characterized the transcriptional arrangement of hscA, showing that it forms part of an operon with the upstream hscB gene, and that it is also expressed from its own promoter. These results are compatible with the P. aeruginosa Hsc66 protein being a functional molecular chaperone involved in the stabilization, in the presence of ethanol, of some proteins required for bacterial growth on short-chain alcohols.
Keywords: Pseudomonas aeruginosa, chaperone, DnaK
The GenBank accession number for the sequence of the W51D chromosomal region including the hscB, hscA and fdxA genes is AF096864.
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INTRODUCTION
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The 70 kDa heat-shock proteins (Hsp70) make up a ubiquitous multigene family of highly conserved proteins. Hsp70 is the most conserved protein known to date which is found in all biota (Gupta & Golding, 1993
). Hsp70 is known to act as a molecular chaperone under normal and stress conditions, stabilizing protein folding intermediates, and to play a major role in thermotolerance (Parsell & Lindquist, 1993
). Eukaryotic organisms have been found to contain multiple Hsp70 family members, while bacteria contain only two (Gupta & Golding, 1993
), and among these, only one, DnaK, has been reported to be functional as a molecular chaperone in vivo. The chaperone activities of DnaK and other Hsp70 chaperones are regulated by DnaJ and Hsp40 accessory proteins, respectively, which stimulate the ATPase activity of the chaperone (Liberek et al., 1991
). The second bacterial DnaK homologue, called Hsc66, is encoded by the hscA gene. The existence of an hscA gene has been documented in the following bacteria: Actinobacillus actinomycetemcomitans (Actinobacillus Genome Sequencing Project, http://www.genome.ou.edu/act.html), Azotobacter vinelandii (Zheng et al., 1998
), Buchnera aphidicola (Clark et al., 1998
), Escherichia coli (Seaton & Vickery, 1994
), Haemophilus influenzae (Fleischmann et al., 1995
), Neisseria gonorrhoeae (Gonococcal Genome Sequencing Project, http://www.genome.ou.edu/gono.html), Neisseria meningitidis (Neisseria meningitidis Sequencing Group at the Sanger Centre, ftp://ftp.sanger.ac.uk/pub/pathogens/nm) and Pseudomonas aeruginosa (Pseudomonas Genome Project http://www.pseudomonas.com). The only case in which it has been shown that the Hsc66 protein actually functions as a molecular chaperone is E. coli, where it has been purified and shown to have a characteristic ATPase activity (Vickery et al., 1997
) and to solubilize protein aggregates (Silberg et al., 1998
). In E. coli it was also shown that the Hsc20 protein, which is homologous to the DnaJ N-terminal domain and is encoded by hscB, functions as its co-chaperone in vitro (Silberg et al., 1998
). The hscB gene is found adjacent to hscA in all the bacteria listed above. In E. coli, the hscBA genes form an operon (Lelivelt & Kawula, 1995
), although there is evidence of promoters transcribing hscA alone (Kawula & Lelivelt, 1994
).
The hscA gene is also found in a wide range of eukaryotic organisms, including yeast, Drosophila and mammals (Konstantopolou et al., 1995
; LéJohn et al., 1994
). In all organisms where the hscA gene is found, its complementary DNA strand presents an open reading frame that could encode a protein homologous to the enzyme glutamate dehydrogenase (Kawula & Lelivelt, 1994
; Konstantopoulou et al., 1995
; LéJohn et al., 1994
). The function of this unusual genetic structure has not been determined. In the case of bacteria, a ferredoxin is encoded downstream of hscA and both genes are transcribed as an operon (Clark et al., 1998
; Fleischmann et al., 1995
; Seaton & Vickery, 1994
; Zheng et al., 1998
); the functionality of this arrangement is also unknown. In A. vinelandii it was suggested that the role of the Hsc66 protein was to stabilize proteins containing an ironsulfur group, such as the one involved in oxygen protection of the nitrogenase enzyme (Zheng et al., 1998
).
In E. coli, hscBA transcription has been shown to be induced by cold shock and not by heat shock (Lelivelt & Kawula, 1995
), but a high level of the Hsc66 protein is maintained under all culture conditions tested, including different incubation temperatures (Lelivelt & Kawula, 1995
). An intriguing observation in this bacterium is that an hscA mutant has no detectable growth defects (Kawula & Lelivelt, 1994
).
Here we describe studies on the ability of an hscA mutant of P. aeruginosa to utilize short-chain alcohols as sole carbon source, and the characterization of the hscA gene.
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METHODS
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Microbiological methods.
Strains and plasmids used in this work are shown in Table 1
. P. aeruginosa strains were grown at 30 °C on LB medium, Pseudomonas Isolation Agar (PIA; Difco) or modified M9 minimal medium (Abril et al., 1989
) supplemented with one of the following carbon sources:glucose 0·2% (w/v), ethanol 1% (v/v). HgCl2 (Hg) was used at a concentration of 12 µg ml-1. Antibiotic concentrations, in µg ml-1, used for P. aeruginosa strain W51D were: carbenicillin (Cb) 50, gentamicin (Gm) 30, kanamycin (Km) 100, tetracycline (Tc) 50. Chloramphenicol was used at 50 µg ml-1 in the experiment to test hscA mRNA induction. For quantification of hscA mRNA, strain W51D was cultivated on LB medium at 30 °C and when the culture reached an OD600 of 0·6, different treatments were applied for an additional 3 h. Alcohols used as carbon sources were used at a concentration of 0·5% (v/v).
Nucleic acid procedures.
DNA isolation, cloning and sequencing, Southern blotting, nick translation and PCR procedures were carried out as described by Sambrook et al. (1989)
. Primer extension analysis was done with primer R3 (see Fig. 1
) located in the 5' region of the hscA gene from P. aeruginosa W51D, using a Thermo Sequenase radiolabelled terminator cycle sequencing kit (Amersham Life Science). Other sequencing reactions were done using Taqfs DNA polymerase and fluorescent dideoxy terminators in a cycle sequencing method; the resultant DNA fragments were separated by electrophoresis and analysed using an automated Applied Biosystems 377 DNA sequencer. To sequence the region flanking the mini-Tn5-Hg insertion in mutant W51M22, oligonucleotides derived from the transposons insertion sequences were used as primers. Total RNA was purified with a DNA-RNA isolation kit (US73750; Amersham Life Science) according to the manufacturers instructions.

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Fig. 1. Nucleotide and protein sequences of the P. aeruginosa W51D hscA regulatory region. The sequence and position of the oligonucleotides used during this work are shown in the figure and identified as Ln or Rn depending on their polarity (L oligonucleotides amplify the sequence from 5' to 3' and R oligonucleotides have the opposite polarity). Position +1 indicates the transcription start site detected which seems to be 70 dependent. RBS, ribosome-binding site sequence for mRNA translation.
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To perform the combined reverse transcriptase (RT) and PCR reactions, total RNA was extracted from a saturated LB culture, and samples were further treated with DNase from Boehringer as instructed by the manufacturer. For the RT reaction 1 µg total RNA was used as template of the RAP2 reverse transcriptase (Amersham) reaction, using 20 pmol of the corresponding primer oligonucleotide (see Fig. 5
). The reaction was incubated for 1 h at 37 °C and inactivated at 70 °C for 5 min. One-tenth of the product of this reaction was used as template for the PCR reaction using the oligonucleotides described in Fig. 5
as templates.

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Fig. 5. Analysis of the hscB and hscA transcriptional arrangement by RT-PCR, according to the procedure described in Methods (see Fig. 1 for the sequences of the oligonucleotides used). Lanes: 1, molecular size markers; 2, RT-PCR performed using R2 and L1 oligonucleotides (amplification of hscBA sequences from a template containing both genes); 3, as in lane 2, but omitting the use of reverse transcriptase; 4, reverse transcription performed using the same oligonucleotides as in lane 2, and PCR performed using R1 and L1 oligonucleotides (amplification of hscB sequences from a template containing both hscB and hscA sequences); 5, as in lane 4, but omitting the use of reverse transcriptase; 6, RT-PCR performed using oligonucleotides R1 and L1 (amplification of hscB sequences only); 7, as in lane 6, but omitting the use of reverse transcriptase.
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Genetic manipulations.
P. aeruginosa matings (Soberón-Chávez et al., 1996
) and transformation (Olsen et al., 1982
) were done as reported previously. The W51D hscA::mini-Tn5-Hg insertion was constructed using the pUT-Hg plasmid (de Lorenzo & Timmis, 1994
). The fdxA::Km mutant was constructed by selection of double recombination events with plasmid pJC9. This plasmid is a derivative of plasmid pJC8, which contains 1223 bp of W51D DNA amplified by PCR using oligonucleotides L2 (5'-GTGCTGCAAGGCGAGCGTGAG-3') and R5 (5'-CAGGCCGGCGACTGG AAATCCCTC-3') (see Fig. 3
), with a Kmr cassette (Alexeyev et al., 1995
) cloned in the StuI site within the fdxA gene. These plasmids include the entire fdxA gene and its flanking sequences. The E. coli hscB, hscA, fdxA operon was subcloned from plasmid pTHK100 (Kawula & Lelivelt, 1994
) into the vector pUCP20 (West et al., 1994
), yielding plasmid pJC10.

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Fig. 3. Molecular characterization of the W51D fdxA mutant W51M23. (a) Schematic representation of the strategy to construct this mutant. (b) Southern blot hybridization of W51D DNA using as probes the W51D fdxA internal fragment (panel I), the W51D chromosomal region cloned in plasmid pJC7 (panel II), or the Kmr resistance cassette (panel III). Lanes in panels II and III correspond to DNAs from: 1, mutant W51M23; 2, W51D; and 3, the plasmid used as probe in each experiment.
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Computer analysis of the DNA and protein sequences.
The sequences were analysed by using the GENE WORKS program (IntelliGenetics) and the University of Wisconsin Genetics Computer Group (UWGCG) programs. The sequences of P. aeruginosa PAO1 contigs were obtained from the Pseudomonas Genome Project website (http://www.pseudomonas.com) released on 15 March 1999.
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RESULTS AND DISCUSSION
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Selection and characterization of mutant W51M22
We have previously reported the selection of a P. aeruginosa strain (W51D) which is able to mineralize at least 70% of a commercial branched-chain alkylbenzene sulfonate mixture and is resistant to high concentrations of these surfactants (Campos-García et al., 1999
; Soberón-Chávez et al., 1996
). In order to determine the degradation pathway of the surfactants, we selected a mutant (W51M22), created by insertion of transposon mini-Tn5-Hg, which was unable to grow on 2-phenylpropionate, a presumed surfactant degradation intermediate. Further characterization showed that neither the parental strain W51D nor the W51M22 mutant was able to use 2-phenylpropionate as a growth substrate; the apparent growth of strain W51D was due to the consumption of ethanol, the solvent used to solubilize 2-phenylpropionate. We concluded that mutant W51M22 was impaired in ethanol utilization (Table 2
) and was not ethanol-sensitive, since it was able to grow in the presence of this alcohol on LB medium at a concentration of 2·5%, the same concentration as tolerated by wild-type strain W51D. The inability of mutant W51M22 to degrade ethanol was studied further (see below).
Sequence analysis of the region adjacent to the transposon insertion
The nucleotide sequence of the W51D genome adjacent to the mini-Tn5-Hg transposon insertion (see GenBank accession no. AF096864) allowed the identification of the hscA gene and, further downstream, apparently forming part of the same transcriptional unit, the fdxA gene (see Fig. 3
). The deduced P. aeruginosa Hsc66 amino acid sequence shows that it belongs to the family of Hsp70 proteins and shows 64% sequence identity with its E. coli homologue.
We found the hscBA and fdxA genes in the PAO1 genome sequence as a part of contig 52 (from nucleotide 445243 to 448125) of the Pseudomonas Genome Project. The sequences of the W51D and PAO1 hscA genes are 91% identical, and both present the antisense ORF encoding a putative glutamate dehydrogenase. In E. coli (Lelivelt & Kawula, 1995
) and A. vinelandii (Zheng et al., 1998
), hscA is the second gene of an operon, with hscB, encoding a DnaJ homologue, being the first transcribed gene. These genes are also clustered in the P. aeruginosa genome and, as shown below, are also transcribed as an operon, although their transcriptional regulation is somewhat different. The PAO1 genomic structure in this region is very similar to that reported for A. vinelandii (Zheng et al., 1998
), containing upstream of the hscB, hscA and fdx gene cluster the iscSUA genes, which are involved in the formation of the ironsulfur cluster of a protein involved in protection of nitrogenase against oxygen damage. P. aeruginosa is unable to fix nitrogen, so the iscSUA genes might participate in the formation of the active site of other proteins containing an FeS cluster.
The mini-Tn5-Hg insertion within the hscA gene was the only insertion of the transposon in mutant W51M22 (Fig. 2
), suggesting that the inability to degrade ethanol was due to inactivation of this gene. This conclusion was supported by the complementation of ethanol utilization by introduction of plasmid pJC7 (Table 1
), containing the W51D hscB, hscA and fdx gene cluster (Table 2
).

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Fig. 2. Molecular characterization of the W51D hscA mutant W51M22. (a) Southern blot hybridization using the mini-Tn5-Hg transposon as probe. Lanes: 1, W51D DNA digested with EcoRI; 24, W51M22 DNA digested with EcoRI (2), BamHI (3) or PstI (4). (b) hscA amplification by PCR of W51D (lane 2) and W51M22 (lane 3) DNAs, using L2 (5'-GTGCTGCAAGGCGAGCGTGAG-3') (Fig. 3 ) and R4 (5'-GCTTATTCCTCGATTTCGTTGAG-3') oligonucleotides. Lanes 1 and 4 correspond to DNA size markers.
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Construction of the fdxA::Tc mutant W51M23
Theoretically a ferredoxin could be involved in the mechanism of alcohol degradation by P. aeruginosa, and the mini-Tn5-Hg insertion within the hscA gene could have a polar effect on the expression of the fdxA gene. To determine whether the W51M22 phenotype was due to the lack of fdxA expression we constructed an fdxA::Km mutant (W51M23) according to the strategy shown in Fig. 3(a)
. Mutant W51M23 had no detectable growth defect (Table 2
); thus the inactivation of hscA seems to be responsible for the inability of mutant W51M22 to use ethanol as a carbon source. Strain W51D possesses multiple ferredoxins as evidenced by Southern blot hybridization using the fdxA gene as a probe (Fig. 3b
, panel I).
Phenotypic characterization of mutant W51M22
The proposed role of the P. aeruginosa Hsc66 protein, based on its homology with other Hsp70 proteins, is the stabilization of protein folding intermediates. We therefore hypothesized that the inability of mutant W51M22 to use ethanol as sole source of carbon was due to the instability in the presence of this alcohol of a protein or proteins absolutely required for ethanol consumption. In accordance with this hypothesis the inability of mutant W51M22 to degrade ethanol was dependent on the alcohol concentration (Fig. 4
).
The enzyme alcohol dehydrogenase, which is required for alcohol degradation, is a candidate for being the critical Hsc66 substrate in this growth condition. To obtain additional evidence on the possible deficiency of an alcohol dehydrogenase activity in mutant W51M22, we determined the ability of this mutant to use different alcohols as growth substrates. Strain W51M22 was unable to grow with C2C4 alcohols, but could readily use decanol and nonanol (Table 2
). These results suggest that an alcohol dehydrogenase which is necessary for short-chain alcohol degradation is deficient in mutant W51M22, due to the lack of Hsc66 activity.
It has been suggested that in A. vinelandii the proteins encoded by the hscA and hscB genes are involved as chaperone and co-chaperone in the maturation of proteins containing ironsulfur clusters (Zheng et al., 1998
). Some alcohol dehydrogenases contain an ironsulfur cluster (Gutheil et al., 1992
). The genetic arrangement of the PAO1 iscSUA and hscB, hscA and fdxA genes is consistent with the proposed role of Hsc66 in the stabilization of FeS-containing proteins.
It was also apparent that the hscA gene product plays a role in the stabilization of proteins involved in ethanol resistance, since the growth of mutant W51M22 on M9 medium supplemented with glucose as carbon source was decreased by the presence of lower concentrations of ethanol than the growth of strain W51D (Fig. 4b
). As mentioned above, the Hsc66 effect on ethanol resistance was not apparent on LB medium.
Complementation of mutant W51M22 by the E. coli hscBAfdx operon
The E. coli Hsc66 and Hsc20 proteins have been shown to have chaperone activity in vitro. In order to obtain additional evidence to support the hypothesis that the W51M22 phenotype was due to the instability of a protein or proteins involved in short-chain alcohol degradation caused by the lack of Hsc66 chaperone activity, we transferred plasmid pJC10, containing the E. coli hscBAfdx operon (Table 1
), to strain W51M22 and determined the complementation of the mutant phenotype.
Strain W51M22/pJC10 was able to grow on C2C4 alcohols and it grew even better than the wild-type strain W51D on ethanol concentrations in the range 23·5% (Table 2
, Fig. 4
). These results strongly suggest that Hsc66 chaperone activity is the limiting factor in the assimilation of short-chain alcohols by W51M22. The presence of plasmid pJC10 renders mutant W51M22 more ethanol resistant than the wild-type strain (Fig. 4b
), suggesting, as mentioned above, that the Hsc66 chaperone also plays a role in ethanol resistance.
Transcriptional regulation of the P. aeruginosa W51D hscA gene
To obtain evidence on the regulatory circuits involved in hscA transcriptional regulation we measured, by RNADNA hybridization in dot-blots, the level of its expression in different culture conditions. hscA transcription was not induced by 3 h treatments with ethanol, heat, cold or chloramphenicol (data not shown). These results show that the E. coli and P. aeruginosa hscA genes are regulated differently, since E. coli hscA has been shown to be induced by cold-shock and chloramphenicol treatment (Lelivelt & Kawula, 1995
). It is interesting, however, that the E. coli hscBAfdxA genes contained in plasmid pJC10 complemented the inability of mutant W51M22 to use ethanol as carbon source (Table 2
, Fig. 4
), thus suggesting that the E. coli and P. aeruginosa Hsc66 proteins have a similar function.
The pattern of expression of the W51D hscB gene is identical to that of hscA as evidenced by the level of its hybridization on RNA dot blots (data not shown), thus suggesting that the two genes form a single transcriptional unit. To further analyse this possibility, we did RT-PCR analysis using as primers oligonucleotides (R1, R2 and L1; Fig. 1
) which would only give a PCR product if hscB and hscA were co-transcribed, yielding a polycistronic mRNA. The results shown in Fig. 5
clearly show the existence of mRNA molecules containing both hscB and hscA sequences.
Primer extension analysis of the region upstream of hscA revealed the presence of a major mRNA start site (Figs 1
and 6
), corresponding to promoters recognized by RNA polymerase containing
70 (Fig. 1
). The appearance of this transcriptional start site was highly reproducible, and could be detected when RNA was extracted from cultures grown at different temperatures or using ethanol as substrate (data not shown). These data show that hscA can be transcribed both from its own promoter and from a promoter upstream of hscB (Fig. 5
). No sequences with homology to known transcriptional activator binding sites were detected in the hscA or hscB 5' regions.

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Fig. 6. Primer extension analysis of the hscA gene using oligonucleotide R3 and the DNA sequence ladder made with the same oligonucleotide. The RNA was extracted from P. aeruginosa W51D grown on LB medium for 16 h.
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Taken together, the results reported herein are compatible with the P. aeruginosa Hsc66 protein being a functional molecular chaperone important for the stabilization in the presence of ethanol of a protein, or proteins, involved in short-chain alcohol degradation. It is tempting to speculate that Hsc66 is specific for the stabilization of proteins containing an FeS cluster, but this hypothesis remains to be experimentally validated.
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ACKNOWLEDGEMENTS
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Jesús Campos-García held a CONACyT scholarship during the development of this work and received support from Programa de Apoyo al Posgrado PADEP (UACPyP/UNAM) project no. 030506. We thank Paul Gaytán, Eugenio López and René Hernández for their technical assistance.
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REFERENCES
|
---|
Abril, M.-A., Michán, C., Timmis, K. N. & Ramos, J. L. (1989). Regulator and enzyme specificities of the TOL plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway.J Bacteriol 171, 6782-6790.[Medline]
Alexeyev, M. F., Shokolenko, I. N. & Croughan, T. P. (1995). Improved antibiotic resistance gene cassettes and omega elements for Escherichia coli vector construction and in vitro deletion/insertion mutagenesis.Gene 160, 63-67.[Medline]
Campos-García, J., Esteve, A., Vázquez-Duhalt, R., Ramos, J. L. & Soberón-Chávez, G. (1999). Branched-chain dodecylbenzene sulfonate degradation pathway of Pseudomonas aeruginosa W51D involves a novel route for degradation of the surfactant lateral alkyl chain.Appl Environ Microbiol 65, 3730-3734.[Abstract/Free Full Text]
Clark, M. A., Baumann, L. & Baumann, P. (1998). Sequence analysis of a 34·7 kb DNA segment from the genome of Buchnera aphidicola containing groEL, dnaA the atp operon, gidA and rho.Curr Microbiol 36, 158-163.[Medline]
Darzins, A. & Chakrabarty, A. M (1984). Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol 159, 9-18.[Medline]
Fleischmann, R. D., Adams, M. D., White, O. & 37 other authors (1995). Whole-genome random sequencing and assembly of Haemophilus influenza Rd. Science 269, 496512.[Medline]
Gupta, R. S. & Golding, G. B. (1993). Evolution of hsp70 gene and its implications regarding relationships between archaebacteria, eubacteria and eukaryotes.J Mol Evol 37, 573-582.[Medline]
Gutheil, W. G., Holmquist, B. & Valle, B. L. (1992). Purification, characterization, and partial sequence of the glutathione-dependent formaldehyde dehydrogenase from Escherichia coli: a class III alcohol dehydrogenase.Biochemistry 31, 475-481.[Medline]
Kawula, T. H. & Lelivelt, M. J. (1994). Mutations in a gene encoding a new Hsp70 suppress rapid DNA inversion and bgl activation, but not proU derepression, in hns-1 mutant Escherichia coli. J Bacteriol 176, 610-619.[Abstract]
Konstantopoulou, I., Ouzounis, C. A., Drosopoulou, E., Yiangou, M., Sideras, P., Sander, C. & Scouras, Z. G. (1995). A Drosophila hsp70 gene contains long, antiparallel, coupled open reading frames (LAC ORFs) conserved in homologous loci. J Mol Evol 41, 414-420.[Medline]
LéJohn, H. B., Cameron, L. E., Yang, B., MacBeath, G., Barker, D. S. & Williams, S. A. (1994). Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by glutamine.J Biol Chem 269, 4513-4522.[Abstract/Free Full Text]
Lelivelt, M. J. & Kawula, T. H. (1995). Hsc66, an Hsp70 homolog in Escherichia coli, is induced by cold shock, but not by heat shock.J Bacteriol 177, 4900-4907.[Abstract]
Liberek, K., Marszalek, J., Ang, D., Georgopoulos, C. & Zylicz, M. (1991). Escherichia coli DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK.Proc Natl Acad Sci USA 88, 2874-2878.[Abstract]
de Lorenzo, V. & Timmis, K. N. (1994). Analysis and construction of stable phenotypes in gram-negative bacteria with Tn5- and Tn10-derived mini-transposons.Methods Enzymol 235, 386-405.[Medline]
Olsen, R. H., DeBusscher, G. & McCombie, W. R. (1982). Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome.J Bacteriol 150, 60-69.[Medline]
Parsell, D. A. & Lindquist, S. (1993). The function of heat shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27, 437-496.
Quandt, J. & Hynes, M. F. (1993). Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria.Gene 127, 15-21.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Seaton, B. L. & Vickery, L. E. (1994). A gene encoding a DnaK/hsp70 homolog in Escherichia coli.Proc Natl Acad Sci USA 91, 2066-2070.[Abstract]
Silberg, J. J., Hoff, K. G. & Vickery, L. E. (1998). The Hsc66Hsc20 chaperone system in Escherichia coli: chaperone activity and interactions with DnaKDnaJGrpE system.J Bacteriol 180, 6617-6624.[Abstract/Free Full Text]
Soberón-Chávez, G., Haïdour, A., Ramos, J. L., Campos, J. & Ortigoza, J. (1996). Selection and preliminary characterization of a Pseudomonas aeruginosa strain mineralizing some isomers in a branched-chain dodecylbenzene sulfonate mixture.World J Microbiol Biotechnol 12, 367-372.
Vickery, L. E., Silberg, J. J. & Ta, D. T. (1997). Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli.Protein Sci 6, 1047-1056.[Abstract/Free Full Text]
West, S. E. H., Schweizer, H. P., Dall, C., Sample, A. K. & Runyen-Janecky, L. J. (1994). Construction of improved Escherichia coliPseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 128, 81-86.
Zheng, L., Cash, V. L., Flint, D. H. & Dean, D. R. (1998). Assembly of ironsulfur clusters. Identification of an iscSUAhscBAfdx gene cluster from Azotobacter vinelandii. J Biol Chem 273, 13264-13272.[Abstract/Free Full Text]
Received 24 September 1999;
revised 10 January 2000;
accepted 17 February 2000.