Functional Complementarity between the HMGl-like Yeast Mitochondrial Histone HM and the Bacterial Histone-like Protein HU*

The mitochondrial histone HM is the major DNA-binding protein in mitochondria and is necessary for maintenance of the mitochondrial genome in the yeast Saccharomyces cerevisiae during growth on fermentable sugars. HM and the Escherichia coli histone-like protein HU have similar activities in vitro, including DNA supercoiling, but share no sequence similarity. We show that HU can functionally complement the respiration deficiency associated with yeast strains lacking HM. Conversely, phenotypes of E. coli cells lacking HU protein, including nucleoid loss and a fila- mentous cell morphology, were alleviated by expression of HM in these cells. The HU protein of bacteria and the HM protein of mitochondria are therefore functionally complementary in vivo. Functional similarities among HM, HU, and the nuclear HMGl pro- teins are implicated and discussed.

The genomes of organisms invariably exist as chromatin (1). Genomic DNA of prokaryotes and eukaryotes is complexed with proteins such as histones, histone-like proteins, and non-histone proteins to form nucleoprotein structures that serve to package and condense the DNA. In the nucleus, the fundamental unit of chromatin is the nucleosome, in which the DNA is wrapped approximately two turns around the histone core (1). The bacterial genome is likewise assembled into a beaded polynucleosome-like structure (Z), and the abundant histone-like protein HU can assemble DNA into nucleosome-like structures in uitro (3)(4)(5). The chromatin structures present in the mitochondrion, however, are poorly characterized.
The mitochondrial histone HM is an abundant basic nuclear-encoded protein in yeast mitochondria that supercoils DNA in uitro (6). HM is not unique to yeast. Homologs have been found in human (7) and amphibian (8) mitochondria. Disruption of the gene encoding HM (ABF2, but referred to here as H I M I ) causes cells to rapidly lose their mitochondrial DNA, resulting in cells that can no longer respire (9). Loss of the HU protein in Escherichia coli is not lethal, but a large percentage of cells in a growing population (11-33%) lose their chromosome, resulting in DNA-less cells (10,ll). Thus, HM is required to maintain the mitochondrial chromosome, and HU is required for maintenance of the E. coli chromosome. Like HM, HU supercoils DNA in uitro in the presence of topoisomerase I, and both proteins are required in a 1:l mass ratio with DNA to obtain maximal DNA supercoiling (3,6). Previous studies in uitro and in vivo indicate that HU * This work was supported by Grant GM43814 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. is important for various cellular functions including DNA replication, transcription, and transposition (5).
According to the endosymbiont theory, the mitochondrion evolved from a free-living aerobic bacterium that formed an intracellular symbiotic relationship with its host, a primordial eukaryote (12). The phylogenetic arrangement of organisms using comparative analysis of ribosomal RNA sequences places the mitochondria in a direct ancestral relationship with purple photosynthetic bacteria, a result that is compatible with other biochemical data (12). This evolutionary relationship combined with similar properties and activities shared by HM and HU in uitro and the requirement for each in chromosome maintenance led us to investigate the functional similarity between the mitochondrial histone HM and the E. coli histone-like protein HU in uiuo. In this study, we show that HU can substitute for HM in yeast mitochondria and that HM can likewise substitute for HU in E. coli. Therefore, although the two proteins share no apparent sequence similarity, HM and HU appear to have similar functions.

EXPERIMENTAL PROCEDURES
Isolation of Mitochondria-Yeast mitochondria were prepared from cultures (300 ml) grown to late-log phase by differential centrifugation of a cell lysate using the procedure of Gasser (13), in which mitochondrial buffer was replaced with 0.25 M sucrose, 1 mM EDTA, 10 mM Pipes,' pH 6.5, and 0.2 mM phenylmethylsulfonyl fluoride.
Purification of HM Protein and NH2-terminal Sequencing-The HM protein was isolated from yeast mitochondria as described (6) and further purified by binding to a Bio-Rex 70 cation-exchange column (Bio-Rad). HM was eluted with 0.45 M NaCl, 10 mM Tris, pH 7.5, 0.1 mM EDTA, and 1 mM dithiothreitol after washing the column with the same buffer containing 0.3 M NaC1. The purified protein was sequenced at the NH, terminus, and the following 21amino acid sequence was obtained KASKRTQLRNE-LIKQGPKRPT.
Cloning of HZMl-The gene encoding the HM protein was cloned from a XZAP yeast genomic library (Stratagene) using an antiserum to the HM protein as a probe. A 2.3-kilobase pair DNA fragment containing the entire coding region of the HM gene was recovered in plasmid form (pHMA1) from the phage clone. This gene was designated HZMl, and DNA sequencing showed that it is identical to the yeast ABF2 gene (9).
Western Blotting-Western blotting was performed essentially as described (14). Antibodies to the E. coli HU protein were a generous gift from Josette Rouvihre-Yaniv. Plasmid Construction-To construct pGAL-HM, the HZMl gene was amplified from yeast genomic DNA by polymerase chain reaction using primers HMPRE (5'-ATGAACAGTTACAGCCTATTAAC-3') and HMA1-9 (5'-CAAAAGCTTGAAGACTTACATAG-3'). This DNA fragment was inserted into the ClaI site of the pRS314G plasmid shuttle vector (TRPl, CENG, and ARS4). pRS314G was a gift from Michael Douglas and contains the 650-base pair GAL1 and GAL10 EcoRI-BarnHI promoter fragment inserted into the EcoRI-BamHI site of pRS314 (15). Plasmid pGAL-HU was constructed by insertion of a DNA fragment containing the HUPA coding sequence (HU2 The abbreviation used is: Pipes, 1,4-piperazinediethanesulfonic acid. protein) fused to the HIMl presequence into the ClaI site of pRS314G as was done for pGAL-HM. The HU2 coding sequence was isolated from genomic E. coli DNA and fused to the HIMl presequence by polymerase chain reaction using primers HU2-1(5'-TTTGGCTAGCand HU2-2 (5'-ACTGCTAGCCAATCTTACTTAACTGCGTC-3').
Plasmid pHIMl contains the HUPA gene promoter (160 base pairs 5' from the initiator methionine) fused to the coding sequence for the mature HM protein (starting from codon 27) in the pSP72 vector (Promega Biotec).
Yeast Methods-The various growth media used in this work are described in Ref. 16. The growth medium YPGalGE (containing yeast, peptone, galactose (0.1%), glycerol, and ethanol) has not to our knowledge been described elsewhere and consists of replacing the 0.1% glucose (dextrose) present in YPDGE (containing yeast, peptone, dextrose (O.l%), glycerol, and ethanol) (16) with 0.1% galactose. Since galactose is a fermentable sugar, like glucose, this medium distinguishes respiration-competent cells from respiration-deficient (petite) cells, as does YPDGE. Disruption of HIMl was accomplished by inserting a 1.1-kilobase pair Hind111 fragment containing the URA3 gene into the NheI site near the NH, terminus of the HIMl coding sequence. One copy of the HIMl gene was replaced with this construct as a linear fragment by one-step gene replacement in yeast strain W303 (17). A diploid strain with one copy of HIMl disrupted (TM144) was sporulated and dissected. Individual asci produced four viable spores, two URA' and two URA-. One haploid segregant that has the HIMl gene disrupted (TM144-15d) and one that is wild-type HIM1 (TM144-15a) were used in this study. tent phenotypes, TM144-15d (him1::URAJ) cells transformed with For the determination of galactose-dependent respiration-compevarious plasmids were grown in galactose-containing liquid medium and then streaked out onto YPDGE and YPGalGE plates and grown at 37 "C, the nonpermissive temperature for growth of himl mutant cells on nonfermentable substrates. The small amount of galactose in YPGalGE was added to induce genes that have been fused to the GAL promoter. The GAL promoter is repressed by glucose, but not by glycerol or ethanol. Yeast strain TM179 was created by mating DNYl (petite lethal) to TM144-9 (himl::URA3). TM179 was sporulated, and isogenic petite lethal (LEU+ and HIS') haploids TM179-5b (HIM1) and TM179-12b (him1::URAJ) differing only at HIM1 were isolated.
Strains-The yeast and bacterial strains used are described in Table I.
Cell Length Measurements-The average lengths of E. coli cells were as determined from negatives of photographs of 4',6-diamidino-2-phenylindole-stained E. coli cells. Several microscope fields and a total of 400 cells/sample were measured using a molecular measurement device (courtesy of Jack Griffith) by projecting the images of cells onto a screen and measuring with a stylus. RESULTS We have purified the HM protein from yeast mitochondria and raised antibodies to it. The protein has DNA supercoiling activity, as reported by Caron et a1 (6). Immunocytochemistry showed that HM is principally localized in mitochondria, indicating that we indeed purified the mitochondrial protein.2 A 2.3-kilobase pair yeast genomic DNA containing the HM coding region was cloned from a yeast genomic library using antibodies as a probe. The deduced amino acid sequence contained the NH2-terminal peptide sequence we obtained from the purified protein, confirming cloning of the HM gene. The gene sequence turned out to be the same as that of ABF2 (autonomous replication sequence-binding factor 2) (9). In this study, we refer to the previously described ABF2 gene as HIMl (histone mitochondria 1). The HM coding sequence contains a 26-amino acid presequence that is not found in the mature HM protein. This appears to be the mitochondrial localization signal sequence. The sequence MNSYSLL-TRSFHESSKPLFNLASTLL was used in this study to target HU and other proteins to the mitochondria.   (17) using the URA3 marker, and a haploid himl (URA') mutant was obtained and shown to lack HM protein. 2 The himl mutant strain (TM144-15d) was examined for its ability to grow on glycerol, where cell growth requires functional (respiring) mitochondria, and on glucose, where functional mitochondria are not required for growth. TM144-15d can be maintained indefinitely on a nonfermentable carbon source like glycerol at 30 "C and therefore is respiration-competent, but grows at about one-third the rate of wild-type HIMl (TM144-15a) cells in liquid glycerol medium at 30 "C. However, at 37 "C, the growth of TM144-15d was completely inhibited in glycerol medium (YPG (containing yeast, peptone, and glycerol)) ( Fig.   lA). TM144-15d is not temperature-sensitive for growth in YPD (containing yeast, peptone, and dextrose (glucose)),2 so the growth defect is specific to mitochondrial function. After growing TM144-15d for multiple generations on fermentable substrates like glucose, where mitochondrial respiration is not required, it would no longer grow in glycerol medium and therefore became respiration-deficient. Yeast cells that have defective mitochondria can be distinguished from respiration-competent cells by growth in medium that distinguishes the mitochondrial mutants as forming small (petite) colonies. This medium, YPDGE, contains glycerol and ethanol as the predominant (nonfermentable) carbon source and a small amount (0.1%) of glucose (dextrose), which enables the respiration-deficient cells to grow, although only enough to form petite colonies. When TM144-15d cells were grown in YPD to late-log phase and plated onto YPDGE, the cell population was predominantly petite, with only a small percentage of cells being capable of forming respiration-competent colonies, as shown in Fig. 1B (himl), corresponding to strains TM144-15a and TM144-15d, respectively. The cells were grown in liquid YPG (glycerol) medium at 30 "C and then streaked onto YPG plates and grown at 30 and 37 "C as indicated. B, the loss of HM protein confers a high frequency of petite phenotype to yeast cells grown on fermentable sugars. Wild-type and himl mutant cells were grown in YPD (glucose) liquid medium (inoculated with a colony from a YPG plate) to late-log phase and plated onto YPDGE medium (see "Experimental Procedures"). Both plates were incubated at 30 "C, and the HIM1 cells were grown for 3 days, whereas the him1 cells were grown for 7 days to allow time for the respiration-competent colonies to grow. Cells that have lost mitochondrial function appear as small (petite) colonies. tent colonies formed by TM144-15d are not revertants because these colonies generatepetites when grown and replated. A growing culture of TM144-15d cells in YPD formed petites at a rate of 5-8%/cell doubling at 30 "C. Cells lacking HM therefore have a high frequency of petite phenotype and are temperature-sensitive for growth on glycerol. We have recently observed the same phenotypes in a strain that is isogenic to TM144-15d, but has the entire coding sequence of HIMl deleted.'

ACCTTGTTGAAGGCTTCCAAGACTCAACTGATTGATG-3')
The himl mutant becomes petite at a high rate due to complete loss of the mitochondrial genome when the cells are not maintained on glycerol, generating rho' mutants (lacking all mitochondrial DNA).' Diffley and Stillman (9) also reported the loss of mitochondrial DNA in abf2 mutant cells.

Complementation of himl Mutant by E. coli HU Protein-
Complementation of cells lacking HM by the E. coli HU protein was investigated by expressing the E. coli HU2 protein from the HUPA gene in the mitochondria of the himl mutant TM144-15d. Fig. 2A shows a schematic diagram of plasmids pGAL-HU and pGAL-HM. These plasmids allow the inducible expression of HM and HU by galactose and their repression by glucose. HU was fused to the HIMl presequence to direct its import to mitochondria (19).3 Mitochondria prepared from TM144-15d carrying pGAL-HU contained the HU protein of the correct size when the cells were grown in the presence of galactose (Fig. 2B). The introduction of pGAL-HM into TM144-15d resulted in the appearance of a high level of HM protein in the mitochondria in the presence of galactose, as shown in Fig. 2B.
To analyze galactose-dependent restoration of respiration competence of the himl mutant by pGAL-HU and pGAL-HM, the TM144-15d cells containing these plasmids were first grown in galactose-containing liquid medium and then streaked onto YPDGE (glucose) and YPGalGE (galactose) plates (see "Experimental Procedures") and incubated at 37 "C, the nonpermissive temperature for growth of TM144-15d when supplied with a nonfermentable carbon source. Fig.  2C shows a limited growth of TM144-15d cells on either plate due to the small amount of fermentable carbon source present. However, the TM144-15d cells containing pGAL-HU grew much better (formed respiration-competent colonies) on galactose than on glucose. Expression of HM from pGAL-HM likewise permitted TM144-15d to form respiration-competent colonies on galactose and not on glucose, whereas TM144-15d cells containing the vector showed no difference in growth on either plate. The pGAL-HU plasmid was less effective than pGAL-HM in reverting the himl growth defect (compared to wild-type cells (labeled HIMl in Fig. 2C)), although the expression of HU clearly enhanced the growth of the TM144-15d strain in the presence of galactose. This discrepancy may be due in part to the difficulty of achieving an optimal level of protein expression in mitochondria. We found that overproduction of HM in wild-type HIMl cells (TM144-15a) or in TM144-15d with pGal-HM results in a high frequency of petite phenotype when cells are grown in the presence of a high concentration (2%) of galactose as opposed to the 0.1% galactose added to YPGalGE.' Maintenance of the mitochondrial DNA is usually not essential for yeast to grow in the presence of fermentable carbon sources such as glucose or galactose. However, yeast strains that contain mutations in the AAC genes (or opl or pet9), which encode the ADP/ATP carrier, are petite lethal, giving rise to nonviable cytoplasmic rhoor rhoo mutants, and therefore must maintain the mitochondrial DNA or die (20, 21). We constructed isogenic petite lethal HIMI+ and himlstrains TM179-5b and TM179-12b, respectively. Neither strain grows on glycerol due to the ouu: mutations, but TM179-5b (HIMI+) grew in YPD at 37 "C, whereas TM179-12b (himl-) did not due to the lack of the HM protein? pGAL-HU rescued the lethal effects of the himl mutation in TM179-12b when the cells were grown on galactose at 37 T ? pGAL-HM also rescued the lethality of TM179-12b at 37 "C as expected, but only on low (0.2%) galactose. pGAL-HM is lethal to TM179-12b and TM179-5b on high (2.0%) galactose? The inability of petite lethal cells lacking HM to grow at 37 "C and their restoration by HU are consistent with the results obtained with TM144-15d shown in Fig. 2.
Another bacterial histone-like protein, the H-NS (or Hla) protein of E. coli, was unable to complement the himl temperature-sensitive growth defect in either TM144-15d or HIMl refers to the mitochondria from wild-type strain TM144-15a, and himl to the TM144-15d strain with the disrupted HIM1 gene. Lanes containing mitochondria from strain TM144-15d (himl) containing the relevant plasmids and grown in the presence of either glucose or galactose (2%) are indicated. The lane labeled E. coli (Wild Type) refers to proteins from the E. coli strain containing wild-type HU genes (YK1100). C, galactose-dependent respiration competence of yeast strains. Strains TM144-15a (HIMI) and TM144-15d (himl) grown in YPGal liquid medium (containing yeast, peptone, and galactose) and strain TM144-15d transformed with vector, pGAL-HM, and pGAL-HU grown in synthetic complement without tryptophan galactose medium to late-log phase were streaked out onto YPGalGE (galactose) and YPDGE (glucose) plates and grown at 37 "C (the nonpermissive temperature). The key indicates the position of each strain on the plates. tein-The restoration of respiration competence to him1 cells by HU led us to investigate the ability of HM to complement an E. coli strain lacking HU protein. Mutations in either HUPA (HU2) or HUPB (HU1) genes in E. coli produce no apparent phenotype. However, null mutations of both genes (hupA,B) result in cells with an extremely filamentous morphology and a high rate of loss of the nucleoid, resulting in DNA-less cells (10, 11).

Complementation of E. coli Cells Lacking HU by HM
We fused the HIMl gene sequence coding for the mature HM protein (without the presequence) to the HUPA gene promoter, and the resulting plasmid (pHIM1) was used to transform the E. coli hupA,B mutant YK1340 (10). The absence of HU and the production of HM in the transformed YK1340 cells were confirmed by immunoblotting (Fig. 3A). YK1340 cells that produced HM protein no longer lost the nucleoid and showed a substantial decrease in the filamentous morphology. Fig. 3B shows the E. coli cells stained with 4',6diamidino-2-phenylindole. The field containing YK1340 cells transformed with the vector demonstrates the characteristic filamentous character of HU-deficient cells, and the arrows point out some of the DNA-less cells that appear as lightly staining bodies (10,ll). In contrast, the YK1340 cells carrying pHIMl maintained their DNA and showed a less filamentous morphology. Fig. 3C illustrates the reduction of the average A.
length of these hup mutant cells from 10.4 pm (8.0 times longer than wild-type cells) to 3.7 pm (2.8 times longer than wild-type cells) due to the expression of HM. The average length of the wild-type cells (YK1100) was 1.3 pm. YK1340 CI -3 -+ + carrying the vector alone produced approximately 11% nu-q z z = .
cleoid-less cells in the growing population compared to very g p g B 4 few DNA-less cells in YK1340 containing pHIMl (<0.3%).

c a
The complementation of hupA,B by pHIMl was observed in all of the original transformant colonies of YK1340 examined in contrast to the transformants obtained with the vector, which consistently exhibited DNA-less cells. Therefore, the results obtained with pHIMl appear not to be due to a possible reversion effect (11) that is unrelated to the presence of pHIM1. We also observed complementation of another hupA,B strain, NH279 (a gift from N. Patrick Higgins), by tial complementation of the hupA,B mutations by the HM protein and show that the yeast HM protein can substitute for HU in E. coli cells with respect to chromosome maintenance. It will be interesting to examine the ability of HM to complement HU-dependent cellular processes such as bactevector

DISCUSSION
We have cloned the gene for the yeast mitochondrial histone HM and have designated the gene HIM1, which is identical to a previously described gene called ABFZ (9). Disruption of HIMl results in cells that lose their mitochondrial DNA at a fast rate on fermentable carbon sources, giving rise to rho' mutants. Furthermore, himl mutants are temperature-sensitive for proper mitochondrial function. In this study, we show that the HM protein and the bacterial histonelike protein HU, which have functional similarities in vitro, can substitute for each other in vivo. HU can revert himl mutants (TM144-15d and TM179-12b) from their temperature-sensitive growth defects and therefore permits mitochondrial function and maintains the mitochondrial DNA despite the absence of HM. Conversely, E. coli cells lacking HU no longer show nucleoid loss when HM protein is present. In addition, H M substantially reduces the filamentous morphology that is characteristic of E. coli cells lacking HU. At this time, the biological function of neither HM nor HU is clearly known. The only common biochemical properties of the two proteins appears to be the DNA supercoiling and the DNA binding activities (6,(22)(23)(24). HM and HU also have in common an essential role in chromosome maintenance in vivo.
It is curious that the disruption of HIMl results in a temperature-sensitive phenotype. Disruption of the PIFl gene, which encodes a mitochondrial helicase, was also shown to cause cells to be thermosensitive for growth on glycerol and to lose their mitochondrial DNA (25,26). It is interesting that the loss of PIF1, a protein that unwinds DNA, a process that induces positive supercoiling of DNA, results in a phenotype that is very similar to that seen for the loss of HM protein, which confers a negative superhelicity to DNA.
Another abundant histone-like chromatin protein in bacteria, the H-NS protein, does not complement the loss of HM in yeast. However, the function of the H-NS protein is apparently very different from that of HU. H-NS appears to be involved in a general DNA "inactivation," being expressed a t higher levels in cells as they reach stationary phase. H-NS is had either the vector or pHIMl had an average length of 1.3 pm. The mutant YK1340 cells containing the vector had an average cell length of 10.4 pm, and the mutant cells carrying pHIMl had an average length of 3.7 pm. required for the regulation of genes in response to environmental changes and does so by mediating changes in DNA topology. However, it does not supercoil DNA itself directly (27,28). In bacteria, some hns mutants show increased plasmid supercoiling (29) and increased Mu transposition (30), unlike hup mutants (HU mutants), which show decreased plasmid supercoiling and decreased Mu transposition (11,31).
The HM protein has a high degree of sequence similarity to the HMGl family of nuclear non-histone proteins (9). The only apparent structural difference between HM and HMGl is the lack of the acid tail domain on the HM protein (32). We have recently demonstrated that the yeast HMGl homolog NHPGA can complement the HM m~t a n t .~ Furthermore, NHPGA can also supercoil plasmid DNA in u i t r~.~ These results are significant and indicate that the function of the HM protein in the mitochondrion is equivalent to the function of HMGl proteins in the nucleus. We are currently investigating the ability of HM to complement the loss of the NHPG genes when targeted to the nucleus. The functional homology between HM and HU and between HM and NHPGA indicates that the functions of HU in E. coli, of HM in mitochondria, and of HMGl in the nucleus are all homologous. This raises an interesting possibility. Perhaps the ancestral HU-encoding gene was lost along with most of the genome inherited by the present-day mitochondrion, and HU was replaced by the functionally similar HM protein, which evolved from a nuclear gene that encodes a protein related to HMG1. Nuclear HMGl proteins may therefore fulfill a role in nuclear chromatin that is similar to the role of HM in mitochondria and of HU in bacteria. Functional similarity between HM and HU is supported by the finding that LEF-1 (a high mobility group box protein) and its cognate DNA-binding site can substitute for the integration host factor (an HU-like protein) and its DNA-binding site in the X phage integrase reaction (33).
Chloroplasts contain a histone-like protein that crossreacts with antisera to HU (18) and may therefore be directly related evolutionarily to HU. The chloroplast HU-like protein might therefore still be encoded by the chloroplast genome.
Thus, it appears that the chloroplast took an alternative evolutionary path than that taken by the mitochondrion, with the chloroplast retaining the HU protein and mitochondria replacing it with HM, a functional homolog.