A Novel H-NS-like Protein from an Antarctic Psychrophilic Bacterium Reveals a Crucial Role for the N-terminal Domain in Thermal Stability
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m211766200
ISSN1083-351X
AutoresChristian Tendeng, Evelyne Krin, Olga Soutourina, Antoine Marin, Antoine Danchin, Philippe Bertin,
Tópico(s)Genomics and Phylogenetic Studies
ResumoWe describe here new members of the H-NS protein family identified in a psychrotrophic Acinetobacter spp. bacterium collected in Siberia and in a psychrophilic Psychrobacter spp. bacterium collected in Antarctica. Both are phylogenetically closely related to the HvrA and SPB Rhodobacter transcriptional regulators. Their amino acid sequence shares 40% identity, and their predicted secondary structure displays a structural and functional organization in two modules similar to that of H-NS in Escherichia coli. Remarkably, the Acinetobacter protein fully restores to the wild-type H-NS-dependent phenotypes, whereas the Psychrobacter protein is no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 °C. Moreover, in vitro experiments demonstrate that the ability of the Psychrobacter H-NS protein to bind curved DNA and to form dimers is altered at 37 °C. The construction of hybrid proteins containing the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein demonstrates the role of the N-terminal domain in this process. Finally, circular dichroism analysis of purified H-NS proteins suggests that, as compared with the E. coli and Acinetobacter proteins, the α-helical domain displays weaker intermolecular interactions in the Psychrobacter protein, which may account for the low thermal stability observed at 37 °C. We describe here new members of the H-NS protein family identified in a psychrotrophic Acinetobacter spp. bacterium collected in Siberia and in a psychrophilic Psychrobacter spp. bacterium collected in Antarctica. Both are phylogenetically closely related to the HvrA and SPB Rhodobacter transcriptional regulators. Their amino acid sequence shares 40% identity, and their predicted secondary structure displays a structural and functional organization in two modules similar to that of H-NS in Escherichia coli. Remarkably, the Acinetobacter protein fully restores to the wild-type H-NS-dependent phenotypes, whereas the Psychrobacter protein is no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 °C. Moreover, in vitro experiments demonstrate that the ability of the Psychrobacter H-NS protein to bind curved DNA and to form dimers is altered at 37 °C. The construction of hybrid proteins containing the N- or the C-terminal part of E. coli H-NS fused to the C- or N-terminal part of the Psychrobacter protein demonstrates the role of the N-terminal domain in this process. Finally, circular dichroism analysis of purified H-NS proteins suggests that, as compared with the E. coli and Acinetobacter proteins, the α-helical domain displays weaker intermolecular interactions in the Psychrobacter protein, which may account for the low thermal stability observed at 37 °C. Life in cold habitats imposes numerous constraints on bacterial metabolism. These conditions require appropriate adaptation of the structure and the physiology of psychrophilic or psychrotolerant bacteria (1Margesin R. Schinner F. J. Biotechnol. 1994; 33: 1-14Crossref Scopus (192) Google Scholar, 2Thieringer H.A. Jones P.G. Inouye M. Bioessays. 1998; 20: 49-57Crossref PubMed Scopus (312) Google Scholar). For instance, the mechanisms allowing these organisms to adapt to low temperature include enhancement of membrane fluidity, which can be obtained through a relative increase in polyunsaturated fatty acids (1Margesin R. Schinner F. J. Biotechnol. 1994; 33: 1-14Crossref Scopus (192) Google Scholar, 3Gounot A.M. J. Appl. Bacteriol. 1991; 71: 386-397Crossref PubMed Scopus (158) Google Scholar). Furthermore, to circumvent the limitations imposed by a reduced thermal energy, enzymatic proteins with a high specific activity are produced (4Gerday C. Aittaleb M. Bentahir M. Chessa J.P. Claverie P. Collins T. D'Amico S. Dumont J. Garsoux G. Georlette D. Hoyoux A. Lonhienne T. Meuwis M.A. Feller G. Trends Biotechnol. 2000; 18: 103-107Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). At the molecular level, all proteins from psychrotrophic organisms studied so far have shown a decrease in their intramolecular interactions, usually associated with both higher flexibility and lower thermal stability as compared with their mesophilic or thermophilic counterparts (5Feller G. Gerday C. Cell. Mol. Life Sci. 1997; 53: 830-841Crossref PubMed Scopus (342) Google Scholar). Most of the data about the molecular adaptation of proteins to low temperature concern psychrophilic enzymes (4Gerday C. Aittaleb M. Bentahir M. Chessa J.P. Claverie P. Collins T. D'Amico S. Dumont J. Garsoux G. Georlette D. Hoyoux A. Lonhienne T. Meuwis M.A. Feller G. Trends Biotechnol. 2000; 18: 103-107Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). In contrast, little is known about regulators of gene expression (6Berger F. Morellet N. Menu F. Potier P. J. Bacteriol. 1996; 178: 2999-3007Crossref PubMed Google Scholar, 7Michel V. Lehoux I. Depret G. Anglade P. Labadie J. Hebraud M. J. Bacteriol. 1997; 179: 7331-7342Crossref PubMed Google Scholar), including nucleoid-associated proteins. The increasing number of sequencing projects has recently revealed the existence of several H-NS-related proteins in Gram-negative bacteria with different life style such as the human pathogenic bacteria Yersinia pestis (8Parkhill J. Wren B.W. Thomson N.R. Titball R.W. Holden M.T. Prentice M.B. Sebaihia M. James K.D. Churcher C. Mungall K.L. Baker S. Basham D. Bentley S.D. Brooks K. Cerdeno-Tarraga A.M. Chillingworth T. Cronin A. Davies R.M. Davis P. Dougan G. Feltwell T. Hamlin N. Holroyd S. Jagels K. Karlyshev A.V. Leather S. Moule S. Oyston P.C. Quail M. Rutherford K. Simmonds M. Skelton J. Stevens K. Whitehead S. Barrell B.G. Nature. 2001; 413: 523-527Crossref PubMed Scopus (976) Google Scholar) and Pasteurella multocida (9May B.J. Zhang Q. Li L.L. Paustian M.L. Whittam T.S. Kapur V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3460-3465Crossref PubMed Scopus (281) Google Scholar) and the plant pathogenic bacteria Xylella fastidiosa (10Simpson A.J. Reinach F.C. Arruda P. Abreu F.A. Acencio M. Alvarenga R. Alves L.M. Araya J.E. Baia G.S. Baptista C.S. Barros M.H. Bonaccorsi E.D. Bordin S. Bove J.M. Briones M.R. Bueno M.R. Camargo A.A. Camargo L.E. Carraro D.M. Carrer H. Colauto N.B. Colombo C. Costa F.F. Costa M.C. Costa-Neto C.M. Coutinho L.L. Cristofani M. Dias-Neto E. Docena C. El-Dorry H. Facincani A.P. Ferreira A.J. Ferreira V.C. Ferro J.A. Fraga J.S. Franca S.C. Franco M.C. Frohme M. Furlan L.R. Garnier M. Goldman G.H. Goldman M.H. Gomes S.L. Gruber A. Ho P.L. Hoheisel J.D. Junqueira M.L. Kemper E.L. Kitajima J.P. Krieger J.E. Kuramae E.E. Laigret F. Lambais M.R. Leite L.C. Lemos E.G. Lemos M.V. Lopes S.A. Lopes C.R. Machado J.A. Machado M.A. Madeira A.M. Madeira H.M. Marino C.L. Marques M.V. Martins E.A. Martins E.M. Matsukuma A.Y. Menck C.F. Miracca E.C. Miyaki C.Y. Monteriro-Vitorello C.B. Moon D.H. Nagai M.A. Nascimento A.L. Netto L.E. Nhani Jr., A. Nobrega F.G. Nunes L.R. Oliveira M.A. de Oliveira M.C. de Oliveira R.C. Palmieri D.A. Paris A. Peixoto B.R. Pereira G.A. Pereira Jr., H.A. Pesquero J.B. Quaggio R.B. Roberto P.G. Rodrigues V. de M.R.A.J. de Rosa Jr., V.E. de Sa R.G. Santelli R.V. Sawasaki H.E. da Silva A.C. da Silva A.M. da Silva F.R. da Silva Jr., W.A. da Silveira J.F. et al.Nature. 2000; 406: 151-157Crossref PubMed Scopus (716) Google Scholar) and Ralstonia solanacearum (11Salanoubat M. Genin S. Artiguenave F. Gouzy J. Mangenot S. Arlat M. Billault A. Brottier P. Camus J.C. Cattolico L. Chandler M. Choisne N. Claudel-Renard C. Cunnac S. Demange N. Gaspin C. Lavie M. Moisan A. Robert C. Saurin W. Schiex T. Siguier P. Thebault P. Whalen M. Wincker P. Levy M. Weissenbach J. Boucher C.A. Nature. 2002; 415: 497-502Crossref PubMed Scopus (726) Google Scholar). Some of these proteins have been studied in detail, e.g. those from Vibrio cholerae (12Tendeng C. Badaut C. Krin E. Gounon P. Ngo S. Danchin A. Rimsky S. Bertin P. J. Bacteriol. 2000; 182: 2026-2032Crossref PubMed Scopus (32) Google Scholar) and Bordetella pertussis (13Goyard S. Bertin P. Mol. Microbiol. 1997; 24: 815-823Crossref PubMed Scopus (28) Google Scholar). All H-NS proteins share the same structural and functional organization in two modules (14Dorman C.J. Hinton J.C. Free A. Trends Microbiol. 1999; 7: 124-128Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 15Bertin P. Hommais F. Krin E. Soutourina O. Tendeng C. Derzelle S. Danchin A. Biochimie (Paris). 2001; 83: 235-241Crossref PubMed Scopus (49) Google Scholar). In enterobacteria, the N-terminal domain of H-NS has been recently shown to contain three α-helices (16Renzoni D. Esposito D. Pfuhl M. Hinton J.C.D. Higgins C.F. Driscoll P.C. Ladbury J.E. J. Mol. Biol. 2001; 306: 1127-1137Crossref PubMed Scopus (32) Google Scholar), whereas the C-terminal three-dimensional structure of the protein resolved by NMR consists of a mix of α-β structures (17Shindo H. Iwaki T. Ieda R. Kurumizaka H. Ueguchi C. Mizuno T. Morikawa S. Nakamura H. Kuboniwa H. FEBS Lett. 1995; 360: 125-131Crossref PubMed Scopus (98) Google Scholar). The H-NS protein forms oligomers via its N-terminal part and is able to bind curved and AT-rich DNA fragments via its C-terminal domain (18Williams R.M. Rimsky S. FEMS Microbiol. Lett. 1997; 156: 175-185Crossref PubMed Google Scholar), both essential properties of H-NS and related proteins (19Bertin P. Benhabiles N. Krin E. Laurent-Winter C. Tendeng C. Turlin E. Thomas A. Danchin A. Brasseur R. Mol. Microbiol. 1999; 31: 319-330Crossref PubMed Scopus (83) Google Scholar). Nevertheless, the role of most proteins of the H-NS family in bacterial physiology remains unknown (15Bertin P. Hommais F. Krin E. Soutourina O. Tendeng C. Derzelle S. Danchin A. Biochimie (Paris). 2001; 83: 235-241Crossref PubMed Scopus (49) Google Scholar). In contrast, in Escherichia coli and Salmonella typhimurium H-NS seem to be involved in bacterial nucleoid organization and in the regulation of various genes involved in adaptation to environmental challenges (20Schroder O. Wagner R. Biol. Chem. 2002; 383: 945-960Crossref PubMed Scopus (79) Google Scholar, 21Hommais F. Krin E. Laurent-Winter C. Soutourina O. Malpertuy A. Le Caer J.P. Danchin A. Bertin P. Mol. Microbiol. 2001; 40: 20-36Crossref PubMed Scopus (341) Google Scholar). A variety of phenotypes has been associated with a mutation in hns, in particular an increase in pH resistance (21Hommais F. Krin E. Laurent-Winter C. Soutourina O. Malpertuy A. Le Caer J.P. Danchin A. Bertin P. Mol. Microbiol. 2001; 40: 20-36Crossref PubMed Scopus (341) Google Scholar, 22De Biase D. Tramonti A. Bossa F. Visca P. Mol. Microbiol. 1999; 32: 1198-1211Crossref PubMed Scopus (221) Google Scholar) and a loss of motility (23Soutourina O. Kolb A. Krin E. Laurent-Winter C. Rimsky S. Danchin A. Bertin P. J. Bacteriol. 1999; 181: 7500-7508Crossref PubMed Google Scholar, 24Soutourina O.A. Krin E. Laurent-Winter C. Hommais F. Danchin A. Bertin P.N. Microbiology. 2002; 148: 1543-1551Crossref PubMed Scopus (54) Google Scholar). Finally, in enterobacteria and related micro-organisms, H-NS proteins are cold shock proteins (12Tendeng C. Badaut C. Krin E. Gounon P. Ngo S. Danchin A. Rimsky S. Bertin P. J. Bacteriol. 2000; 182: 2026-2032Crossref PubMed Scopus (32) Google Scholar, 25La Teana A. Brandi A. Falconi M. Spurio R. Pon C.L. Gualerzi C.O. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10907-10911Crossref PubMed Scopus (241) Google Scholar), which could explain the susceptibility to low temperature of E. coli hns mutant (26Dersch P. Kneip S. Bremer E. Mol. Gen. Genet. 1994; 245: 255-259Crossref PubMed Scopus (85) Google Scholar). To further investigate the structure-function-evolution relationship of H-NS-related proteins, we identified and characterized orthologous proteins in bacteria isolated from extreme environments, i.e. a psychrotrophic bacterium Acinetobacter spp. isolated from Lake Baikal in Siberia 1L. Denissova, unpublished data.1L. Denissova, unpublished data. and a psychrophilic bacterium Psychrobacter spp. collected from Antarctica (27Sun K. Camardella L. Di Prisco G. Herve G. FEMS Microbiol. Lett. 1998; 164: 375-382Crossref PubMed Google Scholar). Like the protein of E. coli, H-NS-like proteins of Psychrobacter and Acinetobacter were both able to complement H-NS-related phenotypes in E. coli at 30 °C. Surprisingly, the Psychrobacter H-NS protein was no longer able to reverse the effects of H-NS deficiency at 37 °C. In vivo and in vitro experiments demonstrated the crucial role of the N-terminal part on the thermal stability of this unusual H-NS protein and give new insight concerning the structural and functional organization of the proteins of this family. Bacterial Strains, Growth Conditions, and Plasmids—The Psychrobacter TAD1 strain was grown at various temperatures (from 4 to 25 °C) in Luria-Bertani (LB) medium. The Acinetobacter bacterial strain 20 was grown from 4 to 37 °C in LB medium. E. coli FB8 strain (28Bruni C.B. Colantuoni V. Sbordone L. Cortese R. Blasi F. J. Bacteriol. 1977; 130: 4-10Crossref PubMed Google Scholar) and BE1410, its hns-1001 derivative (29Laurent-Winter C. Ngo S. Danchin A. Bertin P. Eur. J. Biochem. 1997; 244: 767-773Crossref PubMed Scopus (61) Google Scholar), were used in this study. This H-NS-deficient strain contains a Tn5seq1 transposon insertion located in the 20th codon of the hns gene (30Krin E. Hommais F. Soutourina O. Ngo S. Danchin A. Bertin P. FEMS Microbiol. Lett. 2001; 199: 229-233Crossref PubMed Google Scholar). E. coli cells were grown at various temperatures (from 20 to 37 °C) in LB medium or in M63 medium (31Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 456Google Scholar) supplemented with 40 μg/ml serine, 1 mm isopropyl-1-thio-β-d-galactopyranoside, and 0.4% glucose as a carbon source. Metabolism of β-glucosides was tested on MacConkey agar indicator plates with 1% salicin as a carbon source. Tryptone swarm plates containing 1% Bacto-tryptone, 0.5% NaCl, and 0.3% Bacto-agar were used to test bacterial motility as previously described (23Soutourina O. Kolb A. Krin E. Laurent-Winter C. Rimsky S. Danchin A. Bertin P. J. Bacteriol. 1999; 181: 7500-7508Crossref PubMed Google Scholar). When required, ampicillin or chloramphenicol was added at 100 μg/ml and 20 μg/ml, respectively. Ultracompetent XL1-Blue (Stratagene) cells were used to construct the genomic library of Acinetobacter 20 and Psychrobacter TAD1 strains. All experiments were performed in accordance with the European requirements for the contained use of genetically modified organisms of Group-I (agreement number 2735) and Group-II (agreement 2736 CAI). Plasmid pDIA572 was isolated from a Psychrobacter TAD1 genomic library (see below) and carries a DNA fragment of 1330 bp. The insert nucleotide sequence was determined on both strands. The DNA fragment of pDIA572 contains the hns gene of Psychrobacter TAD1 and its flanking regions (accession number AJ310993). Plasmid pDIA585 carries a 2673-bp DNA fragment containing the hns gene of Acinetobacter strain 20 and its flanking regions (accession number AJ458445). To overproduce the H-NS-His6 proteins of E. coli, Acinetobacter 20, and Psychrobacter TAD1 strains, their structural gene was amplified from genomic DNA using primers 5′-GGAGGTTCATATGAGCGAAGCACTTAAAAT-3′ and 5′-CCGCTCGAGTTGCTTGATCAGGAAATCGT-3′, primers 5′-GGAGGTTCATATGCCAGATATTAGTAATTTATCTG-3′ and 5′-CCGCTCGAGGATGAGGAAGTCTTCCAGTTTCGCACC-3′, and primers 5′-GGAGGTTCATATGACTAATAACACTACTAT-3′ and 5′-CCGCTCGAGTACAGTAAAACTTTCTAGGT-3′, respectively. These pairs of primers introduced a NdeI cloning site and a XhoI cloning site at 5′- and 3′-end, respectively. The PCR products were inserted into the NdeI and XhoI sites of the pET-22b vector (Novagen), giving rise to plasmids pDIA569 (which contains the hns gene of E. coli), pDIA588 (which contains the orthologous gene of Acinetobacter strain 20), and pDIA568 (which contains the orthologous gene of Psychrobacter TAD1). The genes coding for the H-NS chimeric proteins of E. coli and Psychrobacter TAD1 were constructed as follows. The promoter region and the 5′-end of the hns gene of E. coli were amplified from plasmid pDIA547 (19Bertin P. Benhabiles N. Krin E. Laurent-Winter C. Tendeng C. Turlin E. Thomas A. Danchin A. Brasseur R. Mol. Microbiol. 1999; 31: 319-330Crossref PubMed Scopus (83) Google Scholar) using primers 5′-GTTTTCCCAGTCACGAC-3′ and 5′-AGATTTAACGGCAGCAAGGC-3′ and its 3′-end using primers 5′-TAGAAGAGATTTTGAAGGCTGGCACCAAAGCTAAACGTGC-3′ and 5′-AGCGGATAACAATTTCACACAGGA-3′; the 5′-end of the hns gene of Psychrobacter spp. was amplified from plasmid pDIA580 using primers 5′-ATGACTAATAACACTAC-3′ and 5′-AGCCTTCAAAATCTCTTCTA-3′ and its 3′-end using primers 5′-GCCTTGCTGCCGTTAAATCTGGTGAAAGCCTAGAGAAAAAACG-3′ and 5′-CCCAAGCTTGGGTTATACAGTAAAACTTTCTAGG-3′. All constructs were inserted into the HindIII and EcoRI restriction sites of plasmid pDIA547 and gave rise to plasmids pDIA580, pDIA581, and pDIA582 (Table III).Table IIIEffect of temperature on the in vivo complementation of H-NS deficiency in an E. coli hns strain by wild-type and hybrid hns genesView Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Construction of Genomic DNA Libraries—Genomic DNA was isolated from Acinetobacter 20 and Psychrobacter TAD1 bacterial strains. The Psychrobacter genomic library was constructed in plasmid pcDNA 2.1 (Invitrogen) as previously described (15Bertin P. Hommais F. Krin E. Soutourina O. Tendeng C. Derzelle S. Danchin A. Biochimie (Paris). 2001; 83: 235-241Crossref PubMed Scopus (49) Google Scholar), whereas the Acinetobacter genomic library was constructed in plasmid pDIA561 (21Hommais F. Krin E. Laurent-Winter C. Soutourina O. Malpertuy A. Le Caer J.P. Danchin A. Bertin P. Mol. Microbiol. 2001; 40: 20-36Crossref PubMed Scopus (341) Google Scholar). About 60,000 clones were selected on LB plates supplemented with 100 μg/ml ampicillin or 20 μg/ml chloramphenicol and pooled. Large scale plasmid DNA isolation was carried out using the JETstar kit (GENOMED). Protein Purification—The recombinant H-NS-His6 proteins of E. coli and Psychrobacter TAD1 strains were purified from E. coli BL21 (DE3) (Stratagene) carrying pDIA17 and pDIA569 or pDIA568 using NiSO4 chelation columns (Qiagen), as previously described (19Bertin P. Benhabiles N. Krin E. Laurent-Winter C. Tendeng C. Turlin E. Thomas A. Danchin A. Brasseur R. Mol. Microbiol. 1999; 31: 319-330Crossref PubMed Scopus (83) Google Scholar). Gel Retardation Experiments—Gel retardation experiments were performed as previously described (23Soutourina O. Kolb A. Krin E. Laurent-Winter C. Rimsky S. Danchin A. Bertin P. J. Bacteriol. 1999; 181: 7500-7508Crossref PubMed Google Scholar), with H-NS-purified protein of Psychrobacter TAD1 either at 4 or 37 °C. Restriction fragments derived from plasmid pDIA525 that contain flhDC or bla DNA fragments of E. coli were used as competitors. Protein-Protein Cross-linking—Cross-linking experiments were performed as previously described (12Tendeng C. Badaut C. Krin E. Gounon P. Ngo S. Danchin A. Rimsky S. Bertin P. J. Bacteriol. 2000; 182: 2026-2032Crossref PubMed Scopus (32) Google Scholar) with 25 μm H-NS of Psychrobacter TAD1 used in each reaction. After adding cross-linking reagents, i.e. 200 mm 1-ethyl-3(3-dimethylaminopropyl)carbodiimide and 50 mmN-hydroxysuccinimide, the reaction mixtures were incubated for 1 h either at 4 °Corat37 °C, loaded onto a SDS-14% Prosieve acrylamide gel, and silver-stained. Circular Dichroism (CD) Spectroscopy—CD spectra were obtained with a Jobin-Yvon CD6 dichrograph equipped with a thermostatted cell holder. The CD spectra were recorded between 190 and 260 nm from 4 to 70 °C (after 10 min of temperature equilibration before recording the data). The results are the mean values of two successive spectra. CD spectra of purified proteins were determined in pure water with a protein concentration of 14 μm. In Silico Sequence Analysis—The ProtParam software on the Ex-PASy web site (www.expasy.org/tools/protparam.html) was used to determine the amino acid composition of proteins. The CLUSTALw method (32Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (54899) Google Scholar) was used for sequence alignments. Secondary structure prediction was performed using the PREDATOR method (33Frishman D. Argos P. Protein Eng. 1996; 9: 133-142Crossref PubMed Scopus (349) Google Scholar), available on the web site pbil.univ-lyon1.fr. The fold recognition method FROST (Fold Recognition Oriented Search Tool), available on the web site www-mig.jouy.inra.fr/mig/index.html, was used for fold assignments to H-NS protein sequences (34Marin A. Pothier J. Zimmermann K. Gibrat J.F. Tsigelny I. Protein Stucture Prediction; Bioinformatic Approach. International University Line, La Jolla, CA2002: 227-262Google Scholar). Nucleotide Sequence Accession Numbers—The nucleotide sequences of 16 S rRNA and hns genes from Psychrobacter TAD1 bacterial strain have been assigned EMBL nucleotide sequence data base accession numbers AJ310992 and AJ310993, respectively. The 1379 nucleotide sequence of 16 S rRNA gene of Acinetobacter strain 20 was in accordance with the partial sequence in databases under accession number AJ222834. The Acinetobacter spp. hns gene and its product have been assigned EMBL accession number AJ458445. Characterization of Natural Bacterial Isolates from Siberia and Antarctica—Two Gram-negative bacteria were isolated from lake Baikal in Siberia and from frozen water in Antarctica. The first strain was isolated from samples collected from the central basin at about 1,000 m below the surface of the lake (35Soutourina O.A. Semenova E.A. Parfenova V.V. Danchin A. Bertin P. Appl. Environ. Microbiol. 2001; 67: 3852-3859Crossref PubMed Scopus (42) Google Scholar). The selected strain was able to grow under a wide range of temperatures extending from 4 to 37 °C but with an optimum growth temperature of 25 °C. Morphological, biochemical, and phenotypic characterization, e.g. rods occurring in pairs, non-motile bacterium, oxidase-negative and catalase-positive, suggest that this strain belongs to the Acinetobacter genus. Its taxonomic position was further investigated by determination of 16 S rRNA gene sequence (accession number AJ222834) and comparative analysis with different DNA sequences present in databases. The construction of a phylogenetic tree further supports the phylogenetic position of this strain, largely in accordance with morphological characterizations, and suggests that Acinetobacter spp. was closely related to Acinetobacter lwoffii A382 and Acinetobacter johnsonii ATCC 17979 (data not shown). The second strain was collected from frozen continental water in Terre Adélie in Antarctica by C. Gerday, as previously mentioned (27Sun K. Camardella L. Di Prisco G. Herve G. FEMS Microbiol. Lett. 1998; 164: 375-382Crossref PubMed Google Scholar). The morphological and biochemical characteristics of this strain, e.g. coccoid occurring in pairs, oxidase- and catalase-positive, nonmotile bacterium, and growth at low temperatures from 0 to 25 °C with an optimum growth temperature close to 15 °C, indicate that it could be classified either in Moraxella or Psychrobacter groups in agreement with recent data (36Di Fraia R. Wilquet V. Ciardiello M.A. Carratore V. Antignani A. Camardella L. Glansdorff N. di Prisco G. Eur. J. Biochem. 2000; 267: 121-131Crossref PubMed Scopus (24) Google Scholar). Sequence analysis of the 16 S rDNA (accession number AJ310992) together with other characteristics of TAD1 bacterial strain such as a tolerance to 8% NaCl and a susceptibility to bile salt allowed us to refine the classification of this bacterium within the Psychrobacter genus and indicated that this strain could be related to Psychrobacter phenotypic group 2 (phenon 2), which is represented by Psychrobacter uratovorans (37Cavanagh J. Austin J.J. Sanderson K. Int. J. Syst. Bacteriol. 1996; 46: 841-848Crossref PubMed Scopus (162) Google Scholar). Isolation and Characterization of Cold-adapted H-NS-like Proteins—To isolate a putative hns-like gene from both bacterial strains, we took advantage of the serine susceptibility of hns mutants in E. coli (38Lejeune P. Bertin P. Walon C. Willemot K. Colson C. Danchin A. Mol. Gen. Genet. 1989; 218: 361-363Crossref PubMed Scopus (13) Google Scholar). A genomic library was constructed for both strains (see "Materials and Methods"), and each of them was introduced into the hns E. coli strain BE1410. The selection was performed on minimal medium supplemented with serine, as previously described (12Tendeng C. Badaut C. Krin E. Gounon P. Ngo S. Danchin A. Rimsky S. Bertin P. J. Bacteriol. 2000; 182: 2026-2032Crossref PubMed Scopus (32) Google Scholar, 15Bertin P. Hommais F. Krin E. Soutourina O. Tendeng C. Derzelle S. Danchin A. Biochimie (Paris). 2001; 83: 235-241Crossref PubMed Scopus (49) Google Scholar). Several clones were screened at 20 °C for 3 additional phenotypes, i.e. swarming on semi-solid medium, β-glucoside metabolism on MacConkey agar plate, and mucoidy on rich medium. Analysis of the nucleotide sequence of different plasmid DNA inserts revealed the presence of a coding sequence of 321 bp, coding for a 107-amino acid protein with a predicted molecular mass of about 12 kDa and a pI of about 8 in both organisms. The analysis of flanking regions suggests that both genes are not part of a polycistronic operon. As compared with the E. coli H-NS, the modification in the amino acid composition, e.g. the proline content, of the Psychrobacter spp. orthologous protein (Table I) seemed to be similar to that commonly observed from mesophilic to psychrophilic proteins (39Gianese G. Argos P. Pascarella S. Protein Eng. 2001; 14: 141-148Crossref PubMed Scopus (92) Google Scholar). Multiple alignment with various H-NS-related proteins revealed that both cold-adapted proteins share 40% amino acid identity in common; they also showed more than 30% identity with the H-NS-related proteins of Rhodobacter species, i.e. HvrA and SPB, and less than 20% with the E. coli H-NS amino acid sequence (Fig. 1). Despite this, the N-terminal part of these two new proteins was predicted to adopt an α-helical structure, like H-NS in E. coli (data not shown). Moreover, the Acinetobacter H-NS protein displayed the H-NS consensus motif, i.e. YX6(G/S)-(E/D)X(0/2)TW(T/S)G(Q/R)G(R/K)XPX(4/5)AX(3/4)G (0/2, 4/5, and 3/4 indicate 0 or 2, 4 or 5, and 3 or 4 residue(s), respectively) (15Bertin P. Hommais F. Krin E. Soutourina O. Tendeng C. Derzelle S. Danchin A. Biochimie (Paris). 2001; 83: 235-241Crossref PubMed Scopus (49) Google Scholar), whereas the (G/S) residue was replaced by an asparagine in the Psychrobacter protein (Fig. 1). Finally, using FROST, both C-terminal domains were clearly predicted to share a similar three-dimensional structure with the E. coli H-NS protein, with an error rate lower than 1% as indicated by the normalized distances obtained, i.e. 7.5 and 8.1, respectively, for the Acinetobacter and Psychrobacter proteins (34Marin A. Pothier J. Zimmermann K. Gibrat J.F. Tsigelny I. Protein Stucture Prediction; Bioinformatic Approach. International University Line, La Jolla, CA2002: 227-262Google Scholar).Table IAmino acid composition of E. coli, Acinetobacter spp., and Psychrobacter spp.Shown are H-NS proteins using the ProtParam software on the ExPASy web site (www.expasy.org/tools/protparam.html). Modifications in the content of amino acids usually associated with a low protein thermal stability are shown in bold.Amino acid composition of H-NS proteinsE. coliAcinetobacter spp.Psychrobacter spp.Ala (A)15 (11.0%)9 (8.5%)9 (8.5%)Arg (R)11 (8.1%)7 (6.6%)6 (5.7%)Asn (N)6 (4.4%)3 (2.8%)10 (9.4%)Asp (D)6 (4.4%)6 (5.7%)3 (2.8%)Cys (C)1 (0.7%)0 (0.0%)1 (0.9%)Gln (Q)7 (5.1%)8 (7.5%)6 (5.7%)Glu (E)19 (14.0%)11 (10.4%)11 (10.4%)Gly (G)6 (4.4%)5 (4.7%)5 (4.7%)His (H)0 (0.0%)0 (0.0%)2 (1.9%)Ile (I)6 (4.4%)7 (6.6%)8 (7.5%)Leu (L)14 (10.3%)11 (10.4%)10 (9.4%)Lys (K)12 (8.8%)10 (9.4%)9 (8.5%)Met (M)3 (2.2%)0 (0.0%)0 (0.0%)Phe (F)1 (0.7%)2 (1.9%)2 (1.9%)Pro (P)3 (2.2%)4 (3.8%)1 (0.9%)Ser (S)6 (4.4%)9 (8.5%)6 (5.7%)Thr (T)9 (6.6%)4 (3.8%)10 (9.4%)Trp (W)1 (0.7%)2 (1.9%)2 (1.9%)Tyr (Y)3 (2.2%)2 (1.9%)2 (1.9%)Val (V)7 (5.1%)6 (5.7%)3 (2.8%) Open table in a new tab Effect of a Moderate Temperature Increase on in Vivo Biological Activity and in Vitro Biochemical Properties of the Psychrobacter H-NS Protein—Because of the low growth temperature optimum of both Acinetobacter spp. and Psychrobacter spp. strains, the ability of their H-NS-like proteins to complement phenotypes of E. coli hns mutant was evaluated at various temperatures. Remarkably, the overexpression of Acinetobacter protein fully restored, like the E. coli H-NS protein, a wild-type phenotype with regard to β-glucoside utilization and mucoidy at all temperatures tested, whereas the Psychrobacter H-NS protein was no longer able to reverse the effects of H-NS deficiency in an E. coli mutant strain above 30 °C (Table II). This loss of in vivo complementation observed above 30 °C did not result from the proteolysis of the protein. Indeed, the over-expressed H-NS protein of Psychrobacter was visualized on a polyacrylamide gel after extraction from E. coli hns cells grown at 37 °C (data not shown). This observation prompted us to further examine the biochemical properties of purified Psychr
Referência(s)