Gene, 119 (1992) 113-118 0 1992 Elsevier Science Publishers

GENE

B.V. All rights reserved.

113

0378-l 119/92/$05.00

06650

Sequence analysis of a DNA fragment from Buchnera aphidicola (an endosym~iont of aphids) containing genes homologous to drag, rpof), cysE, and se& (Schizaphis graminum; molecular

evolution;

DNA primase;

sigma factor;

serine acetyltransferase;

protein

secretion)

Chi-Yung Lai and Paul B aumann Department of Microbiology, Received by H. Yoshikawa

University of Cal$omia, Davis. CA 95616-8665, 31 March

1992; Revised/Accepted:

USA

30 May]4 June

1992; Received

at publishers:

8 June 1992

SUMMARY

The aphid, ~chizaph~ g~amin~m, contains a prokaryotic, obligately intracellular endosymbiont, Buchnera aph~d~~~la, which is necessary for the survival of the host. A recent study of Bu. aphidicola 16s rRNA has indicated that it is a member of the gamma-3 subdivision of the eubacterial class, Proteobacteria, which includes Escherichia coli. In order to further characterize the endosymbiont and establish its similarity to free-living eubacteria and/or organelles, we have cloned and sequenced a 4534-bp DNA fragment containing dnaG-rpo~-~~~E-~ec3. The deduced amino acid (aa) sequence identity to the homologous E. co/i proteins ranged from 47 to SOo/, . The close proximity of the pair, dnaG-rpo~, to the pair, cy&-s&3, on the Bu. aphidicola DNA, differed from E. coli, in which these two pairs of genes are 14 min apart on the bacterial chromosome. The results of past physiological studies of the endosymbiont were consistent with the presence and function of DNA primase (DnaG), (I factor (RpoD) and components of the secretory system (SecB). Comparison of the deduced aa sequence of Bu. aphidicola CysE (serine acetyltransferase, a key allosterically regulated enzyme in cysteine biosynthesis) with the E. co& wild-type enzyme and a mutant defective in feedback inhibition suggested that the endosymbiont CysE may not be regulated. By analogy with E. co&, the lack of feedback inhibition may lead to overproduction of cysteine by the endosymbiont. The results of this and previous investigations indicate that Bu. aphidicola has many of the properties of free-living bacteria and not of organelles.

INTRODUCTION

Buchnera aphidicola is a prokaryotic endosymbiont of aphids localized in specialized cells (mycetocytes) which

Correspondence to: Dr. P. Baumann, versity of California, Tel. (916) 752-0272

Department

Davis, CA 95616-8665,

of Microbiology,

Uni-

USA.

or -1088; Fax (916) 7529014.

Abbreviations: aa, amino acid(s): B., Baciliirs; Bu., Buchneru; bp, base pair(s); C., C~iurn~~~; CysE, serine acetyltransferase: DnaG, DNA primase;

G+C,

otide(s);

guanine + cytosine;

kb, kilobase

oligo, oligodeoxyribonucleotide;

Pseudomonas;

PCR, polymerase

site(s); RpoD, major wild type.

ORF,

chain reaction;

or 1000 bp; nt, nucleopen reading

frame; P..

RBS, ribosome-binding

sigmafactor; SecB, protein involved in secretion; wt,

form a structure within the body of the aphid known as the mycetome (Douglas, 1989; Ishikawa, 1989; Campbell, 1990; Munson et al., 1991a). The endosymbiont has a Gramcell wall and is enclosed in vesicles derived from the host cell (Houk, 1987). The aphid is dependent on the endosymbiont for survival; treatments which result in the removal of Bu. aphidicola from the host lead to a loss of the reproductive capacity of the aphid. The nature of the contribution which the endosymbiont makes to the survival of the aphid has not been definitively established. Evidence has been presented that the endosymbiont provides the host with a variety of aa (Campbell, 1990; Douglas, 1989; Ishikawa, 1989; Srivastava, 1987). Studies on the evolutionary relationships of Bu. aphidicola by means of sequence analysis of the 16s rRNA have indicated that it is related

114 to E. coli and is a member of the gamma-3 subdivision of the Proteobacteria (Munson et al., 1991b). It has been suggested that the endosymbiont may be a stage in the conversion of a free-living bacterium into a cell organelle (Houk, 1987). In the case of mitochondria, which originated from eubacteria (Woese, 1987) such a change has led to a major loss of genetic information coded by the organelle genome (Taylor, 1983). In order to gain understanding as to the nature of the endosymbiont we have looked for a variety of genes which are present in eubacteria and which have conserved linkage relationships. Bu. aphidicola was found to resemble free-living bacteria in having genes for large and small subunit ribosomal-proteins, initiation factor-3, x-subunit of RNA-polymerase, and proteins involved in DNA metabolism (Munson et al., 1992). An unusual feature has been the finding that the region preceding dnaA (gene for the protein involved in initiation of chromosome replication) lacks characteristic 9-bp sequences known as DnaA-boxes (Lai and Baumann, 1992). DnaA-boxes are highly conserved as is indicated by their presence in a variety of unrelated eubacteria (Yoshikawa and Ogasawara, 1991). In this study we extend our genetic characterization of the endosymbiont by a sequence analysis of a 4534-bp fragment of DNA containing rpoD the gene coding for the major 0 factor of RNA polymerase. In E. coli this gene is part of a complex operon (rpsU-dnaGrpoD) which includes genes for a small ribosomal protein and DNA primase (Burton et al., 1983).

3.3 I& 28kb

dnaG

-

AND DISCUSSION

I

8

rpoD ,

‘!

CVSE SecB,

t

xbxh ,,oow,,,,,,Y 488 bp ,,,a,,.,,,,.*,, 913bp

, (A)

3114

Fig. 1. Genetic map of a 4534-bp line indicates

DNA,

(B)

regions

coding

for proteins,

B,

BumHI; E, EcoRI; S, SpeI; Xb, X&I; Xh, XhoI. All the S and Xh sites are indicated; not all E, B and Xb sites are shown. Thin stripes designate the 488-bp probe used for detection stripes designate XhoI fragment. is presented numerals

indicate

the nt numbering

methods

similar methods endosymbionts

indicate

were described was

1992; Munson by PCR

molec-

et al., 1989); identical

in our publications

amplified

of the ORF;

in Fig. 2. Methods. Standard

(Lai and Baumann,

fragment

whose sequence

the direction

were used (Sambrook

Thick

of the 2.8-kb SpeI-

(A) and (B) indicate two DNA fragments in Fig. 2. The arrows

ular biology

488-bp

of the 3.3-kb EcoRI fragment.

the 913-bp probe used for detection

dealing

or

with aphid

et al., 1991b; 1992). A

using

synthetic

oligos

[5’-

CGGATC CTG CAG AC(TA) TGG TGG AT(TA) (CA)G(TA) CAA GC(TA) AT(TA) AC-3’ BarnHI, &I; 5’-CGG ATC CGT CGA CGC TTT (AT)GC TTC (AT)AT TTG (AT)C(GT) (AT)AT (AT)C(GT) TTC-3’, BumHI, SalI], and then cloned between the PstI and Sal1 sites of pTZ19 (Kunkel et al., 1987). Restriction the endosymbiont presence

DNA,

and Southern-blot

using this construct

of a 3.3-kb EcoRI

DNA was size-fractionated

fragment.

After digestion

by electrophoresis,

and ligated into phosphatase-treated,

as a probe.

EcoRI-cut

iZAP

analyses

of

indicated

the

with EcoRI,

the

electroeluted

from agarose

(Stratagene,

La

Jolla, CA). The 488-bp insert in pTZ 19 was amplified by PCR and s5Slabeled probe was made by nick translation.

AZAP containing

insert was detected

The inserts were excised into

by plaque hybridization.

and subcloned

into pBluescript

vectors (Stratagene).

the 3.3-kb Since the

3.3-kb DNA lacked the beginning of rpoD an additional, upstream DNA fragment was obtained. The 908-bp BarnHI--% fragment was subcloned into pTZ19 and used as a probe in restriction ern blot analysis

(a) General features of the Buchneru uphidicolu DNA fragment The approach used is that of Engel and Ganem (1990) who, based on aa sequences of the major 0 factors (RpoD) of E. coli and B. subtilis, devised a set of oligo primers to two conserved regions of the gene. Using PCR a portion of rpoD was amplified and subsequently used as a probe for the cloning of the full gene from Chlamydia trachomatis, an obligate intracellular parasite which cannot be cultivated outside of animal cells. We used a different set of primers from conserved regions 2 and 4 of Helmann and Chamberlin (1988) for the same purpose (Figs. 1 and 2). Since Bu. aphidicola has a G+C content of 28-30 mol % (Ishikawa, 1987), A+T was used in the third position of degenerate codons. A 488-bp fragment was amplified by PCR (Fig. l), cloned into pTZ19 and used as a probe for the detection of restriction fragments of endosymbiont DNA containing rpoD. A 3.3-kb EcoRI fragment was cloned into lZAP which upon sequence determination was found to be missing the nt sequence coding for the Nterminal portion of RpoD. Using a 913-bp BamHI-XbaI

1

of Bu. aphidicola. Thin

DNA fragment

thick line indicates

=b

E

16175-

phagemids

EXPERIMENTAL

I

E

8

S

was detected.

of endosymbiont

Following

digestion

DNA.

of endosymbiont

Xho I, the DNA was size-fractionated,

enzyme and South-

A 2.8-kb SpeI-XhoI

electroeluted

from the agarose

and ligated into AZAP. The 2.8-kb DNA insert was detected hybridization. the cloning

These and subsequent and sequencing

fragment

DNA by Spe I and gel

by plaque

steps are the same as those used for

of the 3.3-kb

Eco RI fragment

described

above.

fragment as a probe (Fig. 1) a 2.8-kb DNA fragment containing the missing portion was also cloned into AZAP. A genetic map of the Bu. aphidicola DNA fragment is presented in Fig. 1 and the nt sequence in Fig. 2. For convenience the sequence is given as two fragments (Fig. 2A, B) corresponding to the two ORF which are in opposite directions (Fig. 1). Fig. 2A contains nt 1 to 3114 and Fig. 2B nt 1 to 1617. The G+C content of the DNA fragment shown in Fig. 1 is 26.4 mol y0 consistent with the previously cloned fragments (Lai and Baumann, 1992; Munson et al., 1992) and the G+C content of Bu. aphidicoza (Ishikawa, 1987). Comparisons of the sequence of deduced aa with other proteins indicated the presence of four ORF corresponding to a portion of DnaG, and the complete RpoD, CysE, and Set B. In E. coli dnaG and rpoD are part

115 of the rpsU-dnaG-rpoD operon located at 67 min of the E. coli chromosome while cysE and secB are closely linked and located at 81 min (Bachmann, 1990). Bu. aphidicola differs from E. coli in having these four genes in close proximity (Fig. 1). The Bu. aphidicola sequence obtained in this study was deposited in GenBank under the accession No. M90644.

(d) Comparison of sequences involved in regulation In E. coli, P. aeruginosa, A4yxococcus xanthus, Anabaena

(b) Fragment A (dnaG and rpoD) DNA primase is involved in the synthesis of DNA (McMacken et al., 1987). The E. coli enzyme contains 581 aa (Burton et al., 1983). Only a partial nt sequence of Bu. aphidicola dnaG was obtained corresponding to 319 aa

inverted repeats having the characteristics of a Rhoindependent terminator were found in the 174-nt region between rpoD and cysE (Fig. 2A). The presence of inverted repeats in six eubacterial species, some of which are distantly related, and their absence in Bu. aphidicola suggests

(Fig. 2A). The deduced aa sequence was 48% identical to that of E. coli DnaG. The nt sequence of the Bu. aphidicola rpoD and the deduced aa sequence are presented in Fig. 2A. The rpoD was preceded by a potential RBS. The stop codon, UAG, is found at the end of this gene as well as after dnaG. In the ten previously characterized Bu. aphidicola genes UAA was the predominant stop codon (UAA= 8, UGA= 1, UAG = 1) (Lai and Baumann, 1992; Munson et al., 1992). A comparison of the aa sequence of Bu. aphidicola RpoD with that of E. coli and P. aeruginosa (two other members of the gamma-3 subdivision of the Proteobacteria) is presented in Fig. 3 (Burton et al., 1981; Tanaka and Takahashi, 1991). The four highly conserved regions of RpoD noted by Helmann and Chamberlin (1988) are also conserved in the RpoD of Bu. aphidicola; the least conserved sequence is between regions 1 and 2. The aa sequence identity between Bu. aphidicola and E. coli and P. aeruginosa is 80 and 62 %, respectively. The aa identity between E. coli and P. aeruginosa is 66%. These results are consistent with our past studies which indicated a closer relationship of Bu. aphidicola to E. coli than to P. aeruginosa (Munson et al., 1991b).

that some of the regulatory features of rpoD expression in this organism may be different and perhaps reflect an adaptation to a symbiotic association. A similar suggestion was made on the basis of the absence of DnaA boxes (Lai and Baumann, 1992) which have so far been found preceding dnaA of five eubacterial species (Yoshikawa and Ogasawara, 1991). In E. coli the terminal region of dnaG contains a heatshock promoter for the synthesis of RpoD (Neidhardt and VanBogelen, 1987). Sequences resembling this promoter were not found in the corresponding region of the Bu. aphidicola dnaG (Fig. 2A, nt 753 to 794) which also codes for an aa sequence different from that of E. coli. The -10, -35 region of the E. coli cysE promoter has been established (Denk and Bock, 1987); no inverted repeats characteristic of terminators were found in 350 nt downstream from this gene (Tei et al., 1990). The regulation of E. coli secB is not known nor have sequences suggestive of promoters or terminators been detected (Kumamoto and Nault, 1989). In the case of Bu. aphidicola, DNA upstream from cysE and secB did not contain sequences similar to the E. coli cysE promoter or to the E. coli consensus promoter sequence. Similarly, downstream regions did not contain inverted repeats resembling terminators.

(c) Fragment B (secB, cysE) SecB is a component of the protein export system (Kumamoto, 1991; Wickner et al., 1991). The deduced aa sequence of the Bu. aphidicola SecB is 47% identical to the homologous protein of E. coli (Fig. 2B). Gene cysE codes for the enzyme serine acetyltransferase which is part of the pathway of cysteine biosynthesis (Denk and B&h, 1987; Kreditch, 1987). The aa sequence of the Bu. aphidicola enzyme is 52% identical to that ofE. coli. The Bu. aphidicola CysE lacks the 13 C-terminal aa present in E. coli (Fig. 2B). UAA is the stop codon for secB and cysE; both genes are preceded by a potential RBS (Fig. 2B). Both E. coli secB and cysE contain an ORF in the DNA strands opposite to that coding for these two proteins (Kumamoto and Nault, 1989; Tei et al., 1990). No such ORFs were detected in the Bu. aphidicola secB or cysE.

sp., B. subtilis, and C. trachomatis, rpoD is followed by one or two inverted repeats characteristic of Rho-independent terminators (Burton et al., 1983; Wang and Doi, 1986; Inouye, 1990; Koehler et al., 1990; Brahamsha and Haselkorn, 1991; Tanaka and Takahashi, 1991). These inverted repeats begin 7 and 77 nt after the end of rpoD. No

(e) Relation to past physiological studies It has been shown that the closely related endosymbiont of the pea aphid is able to incorporate radioactive precursors into DNA, RNA, and a variety of proteins (Ishikawa, 1989). These results are consistent with the presence of a functioning DnaG and RpoD in the endosymbiont. Bu. aphidicola has a Gramcell wall and by analogy with E. coli its SecB would be expected to be involved in the secretion of proteins destined for the periplasmic space or the outer membrane (Kumamoto, 1991; Wickner et al., 1991). There is evidence indicating that the endosymbiont is the source of cysteine which is utilized by the aphid host (Srivastava, 1987). In E. coli, CysE is synthesized constitutively and the biosynthesis of cysteine is regulated by feedback inhibition of this enzyme by cysteine (Kreditch,

116 (A)

Fig. 2. Nucleotide

sequence

aphidicoiu. (A) nt 1-3114

of the 4534-bp

and (B) nt l-1617

DNA

shown in Fig. 1). The first line is the nt sequence, deduced

aa sequence,

aa sequence indicated

using the designations

E. coli protein.

were made. Vertical line indicates termination

was determined method

the Pharmacia

(Sanger

(Piscataway,

line is the in the

The gene products

arrow, mutational

in the E. coli CysE (Fig. 2B, nt 1420). Potential in (A) indicates

of Eu.

differences

given in Fig. 1. Asterisk,

dash, absence of an aa at that position;

sequence

the second

the third line (if present) indicates

of the homologous

double underline

fragment

(see next page; orientation

are

stop codon; aa substitution

RBS are underlined,

a

region to which the oligo PCR primers insertion

in both directions

of aa in parentheses. using the dideoxy

et al., 1977). Deletions NJ) Nested

Deletion

were obtained Kit. GenBank

The nt chainusing acces-

sion No. M90644.

1987). Denk and Bdck (1987) isolated an E. coli mutant which overproduced and excreted cysteine into the medium. The overproduction was a consequence of a single aa substitution (M+I; Fig. 2B, nt 1482) which resulted in a loss of allosteric inhibition. The Bu. uphidicolu CysE differs greatly from the E. coli protein at the C terminus and it is possible that this difference accounts for a lack of feedback inhibition of the Bu. aphidicola CysE by cysteine. By analogy with the E. coli mutant, this may result in the overproduction of cysteine by the endosymbiont. We are currently testing this possibility by attempting to overproduce the Bu. uphidicolu CysE and determine its allosteric properties.

(B)

Ba EC Pa

M--EHNPKSQLKLL"THGKEPGYLTYREVNDHLPEEIIDSEE

sa EC Pa

APDADDLILNEINTDTDEDAVEAATOVLSSVESELGRTTD IIIlIIlMlAlNTAlEl--IAlIIAlIIIlIIIlIlIIIIIIIIIIlIIIIIlIIIIIII IIIllAlLlIlADTllA--IIIEllAAlAAlIlDIIIlIIllilIIIllIIIIIIIIlII

m

IDIRKRIEEGINQ"QSS"SEYPEAITYLLEOYDRIKTGOI~s~

EC Pa

IIIl1IIIDlIIlIICIIAlIIIlIIIIIIIlNl~~lIIILIIIIIIIIIIlDLAP IElllIIIElIREVMSAIlQFIGTWSIIADIIIIVIlGGlIIIVLSIYIIlDDGSLPlE

M--IQIlQIIIIIIIIRIIIIIIllIlIIIlIlIlDlVlIDIlElIIIlIIIlIIIlMlI MSGKAQQlIRIIEIIAIIRIIIIIllII1lIIIIIIISlPEtVlIIIRIIIIIIlNlFlT [________________~_______ PvRMmmmGTvELLTREGE

I

sa EC Pa

Ba

EC Pa

Ba EC Pa

[........................~~... Ba EC Pa

TIEQ”KDINKRMSIGEAKAKRAKKWEANLRL”ISIAKKYTNRGLQFLDLIQEGNIGLN IlillliiiRIIIIIllItRitlilllllllllilllllllllliiillIIIllllllll IVAEIIEIIIAIIIIIIIlIlllllllllllllllllllllllllIllllllllllllll

Ba EC Pa [_____________________3

. . . . . . . . . . . . . . . . ..~._]

Ba EC Pa

~GREPTPEELSEMLIPEDKIR~L~IARSP~S~E~~IG~~~~S~LG~~IS~~~LEL~L~ MlilllllIIAIRIIMIIIIIiiIIIIlllllliillllllEl/lIlllIIIIIIIIIII ~l11llllilGlllDlltillillllllllllillllllllltiilllllllSlMQSlIE

Ba EC Pa

SATSESLRSATHDVLSGLTAREAKVLRMRFGIDMNTDHTLISA~

[....~.

GAGCCAATTTTATCAAATTTTTATCAARRAA EPILSNFYQKSILNHKKLSH M A s HATL

GTATATTAAATCAT

_____________~~~~~__4_______~_

IIITIIIIAIIIIIIAIlIIIIIIIIIIllll/lllIYIIIIlIIllillllllllllll MIISIIIKESIREIIIIlIIlIIIIlIIIIIIIIIIIHlllllIIlllllllllllllll

AAAAAATTAAGTCAT I

K

EN

G

s

sa EC Pa

ALR~RRPSRSEVLRSFLDD III IlI//IIIIIII/ III II I III IIIHIIIIIIE

III1

Fig. 3. Comparison

617 613 617

of the aa sequence of the RpoD of Bu. uphidicola with

that of two other species of the gamma-3

subdivision

of the Proteobac-

teria. Ba, Bu. aphidicola; EC, E. coli; Pa, P. aeruginosa; dash, absence an aa at that position, ets designate

conserved

vertical line, aa identical regions (Helmann

of

to the aa above; brack-

and Chamberlin,

1988).

linked. In E. coli the pairs dnaG-rpoB and cysE-secB are 14 min apart on the chromosome. (3) Previous physiological studies of the aphid endosymbionts are consistent with the presence and function of DnaG, RpoD, CysE, and SecB in Bu. aphidicola. (4) The absence of inverted repeats characteristic of Rho-independent terminators following rpoD appears to be unique to Bu. aphidicola and suggests a different means of regulation of rpoD expression which may be an adaptation to an endosymbiotic association. (5) The present and past genetic studies of Bu. aphidicola indicate that the endosymbiont resembles free-living bacteria more than it does a cell organelle.

ACKNOWLEDGEMENTS

Conclusions (I) Bu. aphidicola, the endosymbiont of aphids, has genes homologous to E. coli dnaG, I~OD, cysE and secB. (2) In Bu. aphidicola dnaG-rpoD-cysE-secB are closely

(f)

We are grateful to Linda Baumann for the cloning of the 2.8-kb SpeI, XhoI DNA fragment. This research was supported by grant DMB-9201285 from the National Science Foundation and the University of California Agricultural Experiment Station.

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