CURRENT MICROBIOLOGY Vol. 32 (1996), pp. 279–285

An International Journal

R Springer-Verlag New York Inc. 1996

Levels of Buchnera aphidicola Chaperonin GroEL During Growth of the Aphid Schizaphis graminum Paul Baumann, Linda Baumann, Marta A. Clark Microbiology Section, University of California, Davis, CA 95616-8665, USA

Abstract. Buchnera aphidicola is the prokaryotic, intracellular symbiont found in the aphid Schizaphis graminum. Using an immunological approach, we have quantitated the amount of the B. aphidicola chaperonin, GroEL, present in aphid cell-free extracts during the growth cycle of S. graminum at 23°C. Our results indicate that the increase in GroEL approximately follows the increase in aphid weight and endosymbiont number for the first 12 days after birth of the aphid. A 9-day-old aphid contains 1.6 3 105 molecules of GroEL per µm3 of cell volume. This number is similar to that found in Escherichia coli growing at 46°C, close to its maximal growth temperature, and a condition at which there is a major increase in the levels of chaperonins and other stress proteins. It is estimated that at 23°C, 10% of the B. aphidicola protein is GroEL. When S. graminum grown at 23°C was shifted to 33°C for 1 day and subsequently to 23°C, there was no change in the level of GroEL or the rate of growth. It is possible that the high level of GroEL in the endosymbiont masked an increase in the protein owing to a heat shock response.

Survival of aphids (Homoptera: Aphidoidea) is dependent on the presence of bacterial endosymbionts assigned to the genus Buchnera [5, 6, 11, 19, 22]. These organisms are found within aphid cells known as bacteriocytes, which form a bilobed aggregate, the bacteriome, within the body cavity of the aphid. Within the bacteriocytes Buchnera is located in symbiosomes, which are vesicles derived from the bacteriocyte membrane. Both the endosymbiont and the host are dependent on each other. Buchnera has not been cultured outside the aphid host; aphids treated with antibiotics to eliminate endosymbionts are unable to reproduce. In order to gain an understanding of the general physiology and genetics of the association between aphids and their endosymbionts, we have chosen for study the aphid Schizaphis graminum and its endosymbiont Buchnera aphidicola. Our results indicate that this organism has many of the characteristics of free-living bacteria and that Escherichia coli is its closest free-living relative [5, 6]. Plant sap, the diet of aphids, is rich in carbohydrates and deficient in nitrogenous compounds. There is evidence indicating Correspondence to: P. Baumann

that one of the functions of the endosymbionts is the overproduction of the essential amino acids tryptophan [12, 22] and leucine [7], which are required by the aphid host. Bacteria contain a variety of proteins called chaperonins, which are involved in protein folding, translocation of proteins across membranes, and recovery from stress [16, 29]. The level of these proteins can be increased by a variety of physical and chemical agents, including heat (heat shock response). For this reason they are often called ‘‘stress’’ proteins, although all or most of them are essential for the cell under any conditions. Two families of chaperonins are represented by the bacterial proteins GroEL and GroES, which have molecular weights of 57.3 and 10.3 kilodaltons (kDa) respectively [33]. In E. coli the genes for these proteins form one transcription unit which is primarily regulated by the heat shock sigma factor (s32), although some transcription is also mediated by s70, the predominant sigma factor found in cells of this organism during vegetative growth [17, 32]. One of the characteristics of the heat shock response is a major reduction of protein synthesis and the preferential synthesis of heat shock proteins, including GroEL [16]. It has been found that in

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Table 1. Synthetic oligonucleotides used in this study Fusion proteina

Restriction sites

EcMBP-GroEL

EcoRI

EcMBP-GroEL

HindIII

BaMBP-GroEL

EcoRI

BaMBP-GroEL

HindIII

a

nt sequence (58 = 38) GCG TTC GCG GCC GCG TTT GCG CC

GAA GG AAG ACC GAA GG AAG

TTC ATG GCA GCT AAA GAC GTA AAA CTT TCA TTA CAT CAT GCC GCC CAT C TTC ATG GGC GCT AAA GAT GTA AAA CTT ACA TCA TTC CGC CCA TAC CAC

Ba, B. aphidicola; Ec, E. coli; MBP, maltose-binding protein fusion.

Escherichia coli growing at 23°C, 1% of the protein is GroEL (EcGroEL), whereas at 46°C this is increased to 11% [18]. Ohtaka et al. [26] were the first to clone and sequence the groELgroES operon from the endosymbiont of the aphid Acyrthosiphon pisum. Subsequently, the genes of B. aphidicola from the aphid S. graminum were also sequenced and were found to be in the proximity of dnaA, the gene that codes for the protein involved in the initiation of chromosomal DNA replication [5]. Ishikawa [20, 21] has found that aphids treated with cyclohexamide (an antibiotic that prevents host protein synthesis) incorporate radioactive amino acids into GroEL (a protein called symbionin by this investigator). SDS-polyacrylamide gel electrophoresis (PAGE) of endosymbiont extracts, stained for total protein, also indicates the presence of GroEL along with a number of other proteins. It has been shown by immunohistochemistry that in A. pisum GroEL is located in the endosymbionts [14]. Using isolated bacteriocytes and an antibiotic that inhibits host protein synthesis, Aksoy [2] has found that Wigglesworthia glossinidae, the endosymbiont of tsetse flies, preferentially incorporates radioactive amino acids into GroEL. SDS-PAGE of extracts of tsetse endosymbionts indicated that this protein was present in unusually high amounts. Growth of bacteria within eukaryotic cells generally results in an increase in the levels of GroEL [15, 29, 31] (for an exception see [1]). This has been attributed to the generally ‘‘hostile’’ environment of the eukaryotic cell for bacteria. The bacteria respond by increasing the levels of their ‘‘stress’’ proteins, including GroEL. Besides Buchnera and the endosymbiont of tsetse flies, increases in GroEL have also been observed in Rhizobium within plant cells and in the endosymbionts of Amoeba proteus [9]. Although the evidence suggests increased levels of GroEL in the endosymbionts of aphids, most of the conclusions are

based on the high incorporation of radioactive amino acids into GroEL and not the actual amount present [20, 21]. In this study we determined the amount of B. aphidicola GroEL (BaGroEL) present during the growth of S. graminum and, in addition, we determined the effect of a temperature shift on the level of this protein. Materials and Methods pMAL-c2 constructions and purification of fusion proteins. pMAL-c2 is an E. coli expression vector (New England Biolabs, Beverly, Massachusetts, USA) that is used for the construction of fusions between the gene for the maltose-binding protein (MBP) and a gene for a structural protein [3]. The abbreviations used for the fusion proteins are given in Table 1. From the deduced amino acid sequence of BaGroEL (kindly provided by H.K.M. Anwarul, H. Yoshikawa, and N. Ogasawara), it was known that this protein has an 85% amino acid sequence identity to EcGroEL. Past studies have shown that fusions between MBP and B. aphidicola proteins are generally expressed in E. coli at reduced levels compared with fusions of MBP with the homologous E. coli proteins. Consequently we decided to construct and use an EcMBP-GroEL fusion for the preparation of antibody, which would be expected to react well with the endosymbiont protein, owing to the high sequence similarity of EcGroEL and BaGroEL. Similarly, we constructed a BaMBP-GroEL fusion that was used as a standard in the immunological determinations of BaGroEL in aphid cell-free extracts. The synthetic oligonucleotides used for amplification of the appropriate DNA fragment by the polymerase chain reaction [22] are listed in Table 1 together with the restriction enzyme sites used for ligation into pMAL-c2. Overexpression in E. coli of the EcMBP-GroEL and BaMBPGroEL fusion proteins, preparation of cell-free extracts, and affinity chromatography on amylose columns were performed as described [3]. These procedures were followed by an additional purification of the fusion proteins on Q-Sepharose (Pharmacia Biotech, Piscataway, New Jersey, USA) ion exchange chromatography [3]. Antiserum preparation. Two female New Zealand White rabbits, each weighing 2.8 kg, were injected intradermally with 1 mg of EcMBP-GroEL mixed with an equal volume of Freund adjuvant (Difco Laboratories, Detroit, Michigan, USA). Booster doses of 1 mg antigen in an equal volume of incomplete Freund adjuvant were injected intradermally at 5 and 10 weeks. After a further

P. Baumann et al.: Levels of Buchnera GroEL

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6-week interval, the rabbits were given intravenous injections on three alternative days, each injection containing 0.75 mg of the antigen. One week after the last injection, the rabbits were bled by cardiac puncture. Ouchterlony immunodiffusion experiments with EcMBP-GroEL as the antigen indicated that the antisera of both rabbits had approximately the same titer. The antisera were combined, and the immunoglobulins were purified by DEAEAgarose chromatography (Bio-Rad Laboratories, Richmond, California, USA). Following this step, the antiserum was adsorbed with purified MBP and the resulting preparation designated as antiEcGroEL. Growth of aphids and preparation of aphid cell-free extracts. Aphids born over a period of 16–18 h were grown on barley plants as previously described [4]. At different time intervals the aphids were collected, counted, weighed, and suspended to 2% (wt/vol) in a solution containing 52 mM Tris-HCl (pH 6.8), 10 mM phenylthiourea, and 1.7% (wt/vol) SDS [4]. After being ground in a plastic tube with a plastic pestle, the suspension was centrifuged for 30 s in a microcentrifuge at top speed and the supernatant fraction used in SDS-PAGE and Western immunoblots. In experiments in which there was a transient increase in the temperature of the growth chamber, 40–80 aphids were harvested at each time point. Western immunoblots. SDS-PAGE [3] was performed with 0.75 mm, 8% polyacrylamide gels and the Bio-Rad Protean II vertical electrophoresis cell. The proteins were electroblotted onto nitrocellulose by use of the Bio-Rad Trans-Blot cell equipped with a plate electrode. The transfer buffer contained 10% (vol/vol) methanol, and the transfer conditions were 50 V for 20 min at room temperature. Anti-EcoGroEL was used at a dilution of 1/10,000; preimmune sera were used at a dilution of 1/5000. Alkaline phosphatase-conjugated antibody (Promega Corp., Madison, Wisconsin, USA) was used to detect anti-EcGroEL as described by the manufacturer. Intact BaGroEL could not be removed from the BaMBPGroEL fusion by Factor Xa [3] owing to internal cleavage of BaGroEL. Consequently, each Western immunoblot involved the use of known amounts of BaMBP-GroEL as a standard and different amounts or different samples of the BaGroEL-containing aphid cell-free extracts. In experiments in which the amount of BaGroEL was determined at different stages of the aphid life cycle, each SDS-PAGE gel contained duplicates of the following series of samples: three samples of 150 ng BaMBP-GroEL (100 ng BaGroEL equivalents) and protein obtained from 35 µg of aphids harvested at 1, 3, 5, 7, 9, 11, and 12 days. After histochemical visualization of the bands reacting with anti-EcGroEL, the gel was dried for 2–3 h, scanned by means of a Hoefer Scientific Instruments (San Francisco, California, USA) GS-300 scanning densitometer equipped with the GS-370 Data Analysis System, and the peak height determined as previously described [4]. The results presented are based on four replicate determinations. Protein determination. Protein content was determined by the BCA Protein Assay (Pierce Chemical Co., Rockford, Illinois, USA), with bovine serum albumin as the standard.

Results and Discussion Properties of anti-EcGroEL. Figure 1 shows the results of a Western immunoblot containing different protein samples. Anti-EcGroEL did not react with purified MBP (lane a), indicating that all of the

Fig. 1. Western immunoblots with anti-EcGroEL. Lanes: (a) MBP, 30 ng; (b) EcMBP-GroEL, 50 ng; (c) E. coli K-12, 1 µg; (d) extract from 60 µg aphids; (e) BaMBP-GroEL, 250 ng; (f–l) extract from 35 µg aphids harvested at (f) day 1, (g) day 3, (h) day 5, (i) day 7, (j) day 9, (k) day 11, (l) day 12. Underlined numbers are molecular weight standards.

antibody to this protein had been adsorbed (see Materials and Methods). EcMBP-GroEL and BaMBPGroEL each gave single bands which migrated at positions corresponding to 89 and 91 kDa respectively (lanes b and e). (The calculated molecular weight of EcMBP-GroEL is 87.2 kDa and that of BaMPBGroEL is 88.2 kDa.) With E. coli cell-free extracts, only a single band migrating at 58 kDa was detected (lane c; calculated value of EcGroEL is 57.3 kDa). Similarly, Western immunoblots of aphid cell-free extracts showed the presence of a single band migrating at 60 kDa (lanes d, f–l; calculated value of BaGroEL is 58.2 kDa). Determination of the level of GroEL in aphids and E. coli. Previous experiments indicated that S. graminum had the highest number of B. aphidicola and reached maximal weight at 9 days [4]. Consequently, we used 9-day-old aphids for the quantitation of BaGroEL in aphid extracts. Figure 2 shows the results of a densitometric analysis of Western immunoblots in which different amounts of BaMBP-GroEL (expressed as ng BaGroEL equivalents) and aphid extract (expressed as microgram weight of the aphid from which the extract was obtained) were used. The results indicate that 1.0 µg of aphid contains 2.4 ng BaGroEL. We were interested in calibrating our determina-

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Fig. 2. Results of densitometric scans of Western immunoblots containing different amounts of BaGroEL and different amounts of extracts obtained from 9-day-old aphids, expressed as µg of aphids from which the preparation was made. Relative height refers to the height of the peaks obtained in the scans given in arbitrary units.

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Fig. 3. Results of densitometric scans of Western immunoblots containing different amounts of EcGroEL and E. coli K-12. Relative height refers to the height of the peaks obtained in the scans given in arbitrary units. Table 2. GroEL production during the growth of S. graminum

tions by comparisons with other published results. The level of GroEL in E. coli has been determined under a variety of growth rates and temperatures [18, 27]. E. coli growing at 37°C in a glucose minimal medium with a doubling time of 40 min has been shown to contain 1.35% of its proteins as GroEL [27]. Cell-free extracts of E. coli grown under these conditions were prepared, and Western immunoblots containing different amounts of these extracts, as well as different amounts of EcMBP-GroEL (expressed as ng EcGroEL), were performed (Fig. 3). The results of the densitometric analysis indicate that 2.3% of the proteins in the E. coli cell-free extract correspond to GroEL. The reason for the difference between our results and the previously published results [18] is a technical one. Since we were not able to remove intact EcGroEL by Factor Xa from EcMBP-GroEL (see Materials and Methods), we used the fusion protein as the standard. As expected in SDS-PAGE, EcMBP-GroEL migrates a shorter distance than the EcGroEL found in cell-free extracts. Consequently, in Western immunoblots the same amounts of these two proteins would give bands of different widths and intensities. EcMBP-GroEL would have a narrow, intensely stained band, whereas EcGroEL would have a wider and less intensely stained band. Since we measure peak height in the densitometry tracings, the EcMBP-GroEL peak (expressed as EcGroEL equiva-

Number Number of aphids of Days counted samples 1 3 5 7 9 11 12 a

940 410 143 145 172 158 146

7 5 5 5 6 7 6

Aphid weight (µg)

Number of B. aphidicola 3 106/aphida

ng GroEL/ aphid

26.7 6 3.0 65.8 6 1.7 186.0 6 7.6 354.0 6 7.3 422.0 6 9.8 434.0 6 8.0 435.0 6 6.3

0.22 0.53 0.99 4.16 4.75 4.03 4.13

32.8 6 3.2 82.7 6 11.5 242.4 6 26.7 440.5 6 44.0 594.0 6 71.3 563.0 6 90.1 569.5 6 85.7

Data from [4] adjusted for the difference in aphid weight.

lents) would be higher than that of the equivalent amount of EcGroEL in the E. coli cell-free extract. Because of the use of BaMBP-GroEL in the quantitation of BaGroEL in aphid cell-free extracts, the same problem is also found in these determinations. Therefore, all of the values for BaGroEL in aphid cell-free extracts were corrected by multiplying by 0.59 (1.35%/ 2.3%). Comparison of GroEL levels of in B. aphidicola and E. coli. A 9-day-old aphid grown at 23°C weighs 422 µg and contains 4.75 3 106 cells of B. aphidicola and 594 ng of BaGroEL (Table 2). B. aphidicola is approximately spherical with a diameter of 2.5 µm [30]. Using these numbers, we find that B. aphidicola

P. Baumann et al.: Levels of Buchnera GroEL

Fig. 4. Kinetics of increase of aphid-associated Buchnera GroEL during aphid growth. Data for B. aphidicola cell number is from [4] adjusted for the difference in aphid weight.

contains 1.6 3 105 molecules of BaGroEL per µm3 of cell volume. E. coli growing at 37°C in a glucose minimal medium contains 2.35 3 104 molecules of GroEL per cell [8, 27]. Since under these conditions of growth E. coli has a volume of 1.1 µm3 [26], this would correspond to 2.14 3 104 molecules of GroEL per µm3, which is 7.5-fold less than the number of molecules of BaGroEL per µm3 of B. aphidicola. Escherichia coli is able to grow at 46°C, which is close to its maximal growth temperature. Under these conditions 11.9% of its protein is GroEL [18]. Assuming that the concentration of protein per unit volume is the same irrespective of the growth temperature, this would correspond to 1.89 3 105 GroEL molecules per µm3 of E. coli. Therefore, the level of GroEL in B. aphidicola grown at 23°C is slightly less (0.85) than that of E. coli growing near its maximum temperature, under conditions of heat stress, and containing elevated levels of heat shock proteins [18, 27]. If the concentration of proteins per unit volume is the same in both E. coli and B. aphidicola, BaGroEL would constitute 10.1% of the B. aphidicola protein. Levels of GroEL during the growth of S. graminum. Figure 1 (lanes f–l) shows the results of a representative determination of the level of GroEL during growth of the aphid. A summary of all of the determinations is presented in Table 2. A plot of the kinetics of growth of S. graminum and the amount of GroEL and B. aphidicola per aphid is presented in Fig. 4. There is an approximately parallel increase in the weight of the

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Fig. 5. Effect of a transient, 1-day temperature increase on growth and the level of GroEL in the aphid S. graminum. Time indicates days after birth of the aphid. Lanes a–j, Western immunoblots of 35 µg aphid samples harvested at different times. Letters designate bands in Western immunoblots obtained with samples harvested at the time points designated by the same letters. Lane a, 23°C; lanes b–f, 33°C; lanes g–j, 23°C.

aphid and the amount of BaGroEL. At days 11 and 12 there is a slight decrease in GroEL and the number of endosymbionts per aphid. These results are consistent with previous studies indicating an approximately parallel increase of S. graminum weight, B. aphidicola cell number, and B. aphidicola ribosomes during the first 12 days of aphid growth and indicate a close integration of aphid weight increase and endosymbiont number [4, 10]. Effect of a transient 10°C temperature increase on the levels of GroEL. Figure 5 presents the results of an experiment in which S. graminum was growing at 23°C. Four days after birth the temperature of the growth chamber was raised to 33°C for 1 day and subsequently lowered to 23°C for 2 additional days. A temperature of 33°C would be encountered by S. graminum under natural conditions. This temperature was also chosen since transient exposure of A. pisum to 37°C leads to an elimination of Buchnera [24]. At the indicated time points, aphids were harvested, counted, and weighed; cell-free extracts were prepared and the levels of GroEL determined by Western immunoblots. There was no change in the total amount of BaGroEL during the temperature increase or after its decrease (Fig. 5, lanes a–j). Two additional time points at 1 h and 2 h after the temperature shift, which are not

284

included in Fig. 5, gave immunoblot results identical to those of the indicated time points. The shift in the temperature had no significant effect on the growth rate of the aphid (Fig. 5). Visual examination of the aphids did not reveal any deleterious effects of the temperature increase; as in previous growth experiments, newborn aphids were detected at 7 days [4, 10]. Relation of the results to previous studies. The results of our studies show that, as is the case of other intracellular prokaryotes [2, 31], B. aphidicola contains elevated levels of GroEL similar to those found in E. coli growing at its maximal temperature [18]. During the growth of the aphid, the level of GroEL approximately parallels the increases in the weight of the aphid, the B. aphidicola cell number, and the B. aphidicola ribosome content [4, 10]. In addition, the level of BaGroEL remains unchanged during a 1-day shift from 23°C to 33°C (Fig. 5). The amount of GroEL protein found in B. aphidicola is consistent with the amount of GroEL found in SDS-PAGE of total proteins of endosymbiont from the aphid A. pisum [20]. It appears to be considerably less than the amount of GroEL found in tsetse fly primary endosymbionts [2]. Studies of the heat shock response differentiate between the increased rate of incorporation of radioactive amino acids into heat shock proteins and the total level of the proteins. In E. coli, upon temperature shock, there is an increase in the incorporation of radioactivity into GroEL followed by a new and higher steady state level of this protein [16]. In Chlamydia trachomatis there is an increase in the incorporation into GroEL, but the steady state level remains the same [13]. It has been shown that purified Buchnera incubated at 33°C incorporates more radioactivity into GroEL compared with endosymbionts incubated at 20° or 25°C [23]. These results, together with the results of our investigations, resemble those found with C. trachomatis. Upon an increase in temperature there was evidence for a heat shock response, as measured by incorporation of radioactive amino acids, but no detectable increase in the steady state level of GroEL. The explanation for these observations probably resides in the fact that B. aphidicola already contains a large amount of GroEL, and the transient increase in the synthesis of this protein is masked by its high level. Sequence comparisons in Buchnera from different species of aphids have been useful for the identification of putative promoters, since these regulatory regions are conserved [5]. Comparisons of the regions upstream of groEL of the endosymbionts from both A.

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pisum and S. graminum indicated that the only sequences conserved resembled those recognized by s32, a sigma factor involved in the heat shock response [17, 33]. Since the level of GroEL in B. aphidicola is high, it is probable that this sigma factor is always functional and is solely responsible for the high levels of this protein. It has been claimed that Buchnera in the aphid synthesizes GroEL as its primary or only protein [20, 21]. The evidence against this interpretation has been recently discussed [5]. Our results do indeed indicate an unusually high level of BaGroEL, but do not support the conclusion that it is the sole or primary protein made by the endosymbiont. B. aphidicola has been found to have many of the genes for DNA replication, transcription, translation, amino acid biosynthesis, protein secretion, and intermediary metabolism [5]. Its growth is integrated with that of the aphid host [4]. Consequently, it must have the enzymes, proteins, and ribosomes necessary for orderly bacterial growth, and they must function in the Buchnera cells located within the aphid host. Incorporation of radioactive precursors into DNA, RNA, and proteins has also been demonstrated in purified endosymbionts [21]. The term ‘‘stress proteins’’ or ‘‘heat shock response’’ embody useful concepts when applied to many organisms subjected to a harmful stimulus. The transient nature of the stimulus and a comparison with some ‘‘normal’’ basal state is also implicit in these terms. These designations lose their conceptual usefulness when applied to organisms that have high levels of stress proteins and live in and are adapted to habitats that are not the usual sources from which common organisms are isolated, and are also habitats that do not resemble the laboratory conditions under which most organisms are routinely grown. The Buchnera-aphid association is 200–250 million years old, and it is conceptually misleading to state that owing to the high level of GroEL the endosymbionts are stressed [23], since the aphid is their normal habitat to which they are adapted. Finally, it should be mentioned that van den Heuvel et al. [28] have recently presented evidence that GroEL is found in the aphid hemolymph, although the actual amount is not known. Since Fukatsu and Ishikawa [14] have found by immunohistochemistry with anti-GroEL antibody that the endosymbionts alone stain intensely, and since GroEL has no export sequences, it is probable that this protein in hemolymph comes from lysed Buchnera cells. Since there is no evidence of extensive cell lysis during active growth of the aphids [5], the level of GroEL in the

P. Baumann et al.: Levels of Buchnera GroEL

hemolymph probably represents a relatively small fraction of the total GroEL and should not significantly affect the results of our calculations of the level of GroEL in Buchnera cells. ACKNOWLEDGMENTS This material is based on work supported by the National Science Foundation under award no. MCB-9402813 and by the University of California Experiment Station. We thank H.K.M. Anwarul, H. Yoshikawa, and N. Ogasawara for making available the sequence of B. aphidicola GroEL from the aphid S. graminum.

Literature Cited 1. Abshire KZ, Neidhardt FC (1993) Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage. J Bacteriol 175:3734–3743 2. Aksoy S (1995) Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin. Insect Mol Biol 4:23–29 3. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) (1995) Current protocols in molecular biology. New York: Wiley 4. Baumann P, Baumann L (1994) Growth kinetics of the endosymbiont, Buchnera aphidicola, in the aphid Schizaphis graminum. Appl Environ Microbiol 60:3440–3443 5. Baumann P, Baumann L, Lai CY, Rouhbakhsh D, Moran NA, Clark MA (1995a) Genetrics, physiology, and evolutionary relationships of the genus Buchnera: intracellular symbionts of aphids. Annu Rev Microbiol 49:55–94 6. Baumann P, Lai CY, Baumann L, Rouhbakhsh D, Moran NA, Clark MA (1995b) Mutualistic association of aphids and prokaryotes: biology of the genus Buchnera. Appl Environ Microbiol 61:1–7 7. Bracho AM, Martı´nez-Torres D, Moya A, Latorre A (1995) Discovery and molecular characterization of a plasmid localized in Buchnera sp. bacterial endosymbiont of the aphid Rhopalosiphum padi. J Mol Evol 41:67–73 8. Bremer H, Dennis PP (1987) Modulation of chemical composition and other parameters of the cell by growth rate. In Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella tryphimurium; Cellular and Molecular Biology. Washington DC: American Society for Microbiology, pp 1530 9. Choi EY, Ahn GS, Jeon KW (1991) Elevated levels of stress proteins associated with bacterial symbiosis in Amoeba proteus and soybean root nodule cells. BioSystems 25:205–212 10. Clark MA, Baumann L, Baumann P, Rouhbakhsh D (1996) Ribosomal protein S1(RpsA) of Buchnera aphidicola, the endosymbiont of aphids: characterization of the gene and detection of the product. Curr Microbiol 32:89–94. 11. Douglas AE (1989) Mycetocyte symbiosis in insects. Biol Rev Camb Philos Soc 64:409–434 12. Douglas AE, Prosser WA (1992) Synthesis of the essential amino acid tryptophan in the pea aphid (Acyrthosiphon pisum) symbiosis. J Insect Physiol 38:565–568 13. Engel J, Pollack NJ, Perara E, Ganem D (1990) Heat shock response of murine Chlamydia trachomatis. J Bacteriol 172: 6959–6972

285 14. Fukatsu T, Ishikawa H (1992) Synthesis and localization of symbionin, an aphid endosymbiont protein. Insect Biochem Mol Biol 22:167–174 15. Garbe TR (1992) Heat shock proteins and infection: interactions of pathogen and host. Experientia 48:635–639 16. Georgopoulos G, Ang D, Maddock A, Raina S, Lipinska B, Zylicz M (1990) Heat shock response of Escherichia coli. In Drlica K, Riley M (eds) The bacterial chromosome. Washington DC: American Society for Microbiology, pp 405–419 17. Gross CA, Straus DB, Erickson JW, Yura T (1990) The function and regulation of heat shock proteins in Escherichia coli. In Morimoto RI, Tissie`res A, Georgopoulos C, (eds) Stress proteins in biology and medicine. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp. 167–189 18. Herendeen SL, vanBogelen RA, Neidhardt FC (1979) Levels of major proteins of Escherichia coli during growth at different temperatures. J Bacteriol 139:185–194 19. Houk EJ, Griffiths GW (1980) Intracellular symbiotes of the Homoptera. Annu Rev Entomol 25:161–187 20. Ishikawa H (1984) Molecular aspects of intracellular symbiosis in the aphid mycetocyte. Zool Sci 1:509–522 21. Ishikawa H (1989) Biochemical and molecular aspects of endosymbiosis in insects. Int Rev Cytol 116:1–45 22. Lai C-Y, Baumann L, Baumann P (1994) Amplification of trpEG: adaptation of Buchnera aphidicola to an endosymbiotic association with aphids. Proc Natl Acad Sci USA 91:3819– 3823 23. Morioka M, Ishikawa H (1992) Mutualism based on stress: selective synthesis and phosphorylation of a stress protein by an intracellular symbiont. J Biochem 111:431–435 24. Ohtaka C, Ishikawa H (1991) Effects of heat treatment on the symbiotic system of an aphid mycetocyte. Symbiosis 11:19–30 25. Ohtaka C, Nakamura H, Ishikawa H (1992) Structures of chaperonins from an intracellular symbiont and their functional expression in Escherichia coli groE mutants. J Bacteriol 174:1869–1874 26. Pang H, Winkler HH (1994) The concentration of stable RNA and ribosomes in Rickettsia prowazekii. Mol Microbiol 12:115– 120 27. Pedersen S, Bloch PL, Reeh S, Neidhardt FC (1978) Patterns of protein synthesis in E. coli: a catalog of the amount of 14 different proteins at different growth rates. Cell 14:179–190 28. van den Heuvel JFJM, Verbeek M, van der Wilk F (1994) Endosymbiotic bacteria associated with circulative transmission of potato virus by Myzus persicae. J Gen Virol 75:2559– 2565 29. Watson K (1990) Microbial stress proteins. Adv Microb Physiol 31:183–223 30. Whitehead LF, Douglas AE (1993) Population of symbiotic bacteria in the parthenogenetic pea aphid (Acyrthosiphon pisum) symbiosis. Proc R Soc Lond Ser B Biol Sci 254:29–32 31. Young DB, Mehlert A, Smith DF (1990) Stress proteins and infectious diseases. In Morimoto RI, Tissie`res A, Georgopoulos C (eds) Stress proteins in biology and medicine. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, pp 131–165 32. Yura T, Nagai H, Mori H (1993) Regulation of the heat-shock response in bacteria. Annu Rev Microbiol 47:321–350 33. Zeilstra-Ryalls J, Fayet O, Georgopoulos C (1991) The universally conserved GroE(Hsp60) chaperonins. Annu Rev Microbiol 45:301–325