Oene, 94 (1990) 69-75 Elsevier

69

GENE03~2

Cloning, sequence and expression in E s c h e r i c h i a coil of the M e t h y l o b a c U l u s f l a g e l l a t u m

recA gem

(Obfigate methylotroph; recombinant DNA; general recombination; DNA repair; prophage induction; SOS box; LexA repressor; phasmids)

Mark Gomelsky, Eugene Gak, ,Audrey Chistoserdov, Alexander Bolotin and Yuri D. Tsygankov Institute of Genetics and Selection of lndus~ai Microorganisms, Moscow 113343 (U.S.S.R.) Received by J. Davidson: 6 March 1990 Accepted: 10 May 1990

SUMMARY

By means of interspecific complementation of an Escherichia coli recA - mutation with phasmids containing a gene bank from an obligate methylotroph, Methyiobacillusflagellatum (Mf), the recA + gene from this bacterium was identified. When expressed in an E. coil recA - host, it can function in recombination, DNA repair, and prophage induction. The nucleotide sequence of the gene has been determined. The coding region consists of 1032 bp specifying344 amino acids. The deduced RecA protein structure shows a striking homology with RecA from other bacteria, except for the C-terminal region and some residues which were proposed to be responsible for the coprotease ability of RecA proteins. The region preceding the recA-Mf gene start codon has no SOS box - the LexA repressor binding site. Expression of the recA-Mfgene in E. coil proved to be DNA-damage independent.

INTRODUCTION

The r e ~ + gene product acts in several processes involving DNA metabolism. For E. coli it has, been demonstrated to participate in general recombination, .DNA repair, mutagenesis and expression of SOS functions. The Correspondence to: Dr. Y.D. Tsygankov, Institute of Genetics and Selection of Industrial Microorganisms, l-st Dorozhny pr-d, l, Moscow 113545 (U.S.S.R.) Tel. 315-37-74. Abbreviations: aa, amino acid(s); Ap, ampiciilin; bp, base pair(s); £c, Escherichia coil;pGai, p-galactosidase; ,4, deletion; IPTG, isopropyi-p-vthiogalactopyranoside; kb, kilobase(s) or 1000bp; LB, Luria-Bertani (medium); LexA, common repressor for SOS network; MC, mitomycin C; Mr, Methylobacillusflagellatum; MMS, methyl methane suifonate; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; Pa, Pseudomonas aeruginosa; Pm, Proteusmirabilis; pfu, plaque-forming units; Pollk, Klenow (large) fragment of E. coil DNA polymerase I; R, resistance/resistant; s, sensitivity/sensitive; SD, ShineDalgarno; SDS, sodium dodecyl sulfate; SOS box, LexA binding site; Sy, Synechococcus; UV, ultraviolet light; XGai, 5.bromo-4-chloro-3-indolylp-D-galactopyranoside; [ ], denotes plasmid carrier state; (), denotes prophage (lysogenic) state; ::, novel joint. 0378-1119/90/$03.50@ 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)

E. coli RecA (RecA-Ec) protein combines several enzyme

activities, the most studied are: ATP-dependent recombinase and coprotease activities (Little and Mount, 1982; Walker 1984; Lanzov, 1985). The former is essential for processes including homologous recombination, and the latter for SOS network derepression and promoting of cleavage of some phage repressors. The ability to promote the proteolysis of phage repressors and a common repressor for SOS genes - LexA protein (Little, 1984; Slilaty and Little, 1987) - is caused by an as yet unidentified signal that appears as a response to DNA damage and blocked replication. Recently, other RecA activities have been demonstrated (Foster and Sullivan, 1988; Nohmi et al., 1988; Vericat et al., 1988). A protein analogous to RecA-Ec has been shown to exist in a variety of bacterial strains other than E. coil K-12 (Eitner etal., 1981; Better and Helinski, 1983; Keener et al., 1984; Ohman et al., 1985; Kokjohn and Miller, 1985; Zaitzev et al., 1986; Sano and Kageyama, 1987; Goldberg and Mekalanos, 1986; Hamood et al., 1986; Paul et al., 1986; Finch etal., 1986; Koomey and Falkow, 1987;

70 Goodman et al., 1987; Resnick and Nelson, 1988; Ramesar et al., 1988). The analysis of various properties bacterial RecA proteins and the structures of some of them (Horii et al., 1980; Sancar et al., 1980; Kawashima et al., 1984; Sano and Kageyama, 1987; Murphy et al., 1987; Akaboshi et al., 1989; Ramesar et al., 1989) revealed a striking degree of similarity. Several domains responsible for various RecA-Ec activities have been determined (Kawashima et al., 1984; Wang and Tessman, 1986; Ogawa and Ogawa, 1986), This has made possible the comparison of RecA structures and properties. The regulation of the recA-Ec gene expression as well as regulation of other participants of the E. coli SOS network is dependent on the LexA repressor which binds to the specific nt sequence in the regulatory regions of SOS genes (Little and Mount, 1982; Walker, 1984). Most of the bacterial recA + genes, cloned in E. coli, seem to be inducible by DNA-damaging agents that supposes the regulation of their transcription by LexA-Ec. In some cases induction has not been observed (Finch et al., 1986; Goodman et al., 1987; Ramesar et al., 1989), perhaps because ofthe absence ofan SOS box in the recA ÷ operator regions. It means that essential differences may occur in the performance of SOS systems of various organisms. The aim of the present study was the cloning, characterization, sequencing, and expression in E. coli of the recA + gene from the obligate methanol utilizer M.flagellatum (Kletsova et ai., 1987). The biochemical potential and some technological advantages make this bacterium a prospective subject for prokaryotic and eukaryotic expression applications (Chistoserdov et al., 1987).

RESULTS AND DISCUSSION

(a) Identification of the Methylobacillus flagellatum recA + gene

The gene bank of M.flagellatum was constructed by cloning of EcoRI fragments of M.flageilatum DNA into phasmid ~pMYFI31 (Yankovsky etai., 1989). Recombinant phasmids were maintained in E. coli HBI01(~)recA- strain (Maniatis et al., 1982) on LB agar (Maniatis et al., 1982) + 50 #g Ap/ml. Several clones were revealed by their ability to grow on LB agar supplemented with 0.3 #g MC/mi. Resistance to the DNA-damaging agent MC, which is lethal for recAstrains, could be due to the complementation of HBI01 recA - mutation by the cloned fragment. Interspecific complementation of the repair deficiency of E. ccli recAstrains is a traditional approach for foreign recA + genes search in the clone libraries. Recombinant phasmids that restored the MC R phenotype of E. coli were digested with EcoRI. All of them

contained four identical fragments of 8.5, 2.6, 1.8, and 1.0 kb. One (pREC1) was used for further study (Fig. 1). When pREC 1 was re-transformed into HB lOlrecA- cells, all the transformants demonstrated resistance to MC, confh-ming that MC R phenotype was provided by pREC1 rather than from HB101 reversion to recA +. Restriction analysis and localization of the recA-Mfgene in the cloned fragment are shown in Fig. 1. (b) Restoration of the recombination, D N A repair, prophage induction in Escherichia coli recA - cells by r e c A . M f gene product Compensation of the recombination defect in E. coli recA- strain by pREC1 was demonstrated in P1 trans-

duction experiments. The results presented in Table I show that pREC1 restored approx. 50~ of the recombination

~g

l~g l~g

.8{S

l

2.6 " I

,

,

,~

. ~g ~ • ' ' ' " " .... -'"4~0"

E Z.6

, pRECl

l~'""., pRF.C~

p.,"

1.0 kb

4

E47

E pREC6

Fig. !. Restriction map and localization of the recA.M/'gene.containing fragment, pRECI is ~.pMYFI31 phasmid (Yankovsky et al., 1989) with 13.9-kb insertion of M.flagellatum DNA into EcoRl site. It conferred MC R phenotype to HBi01 recA- strain, pREC5 was obtained by the subcioning of corresponding BamHI-Bglll fragment into the BamHl site ofpUCi9 (Yanisch-Perron et al., 1985). pREC6 is a derivative ofpREC$, the heavy line corresponds to the recA.Mf, which was localized by deletion analysis. Restriction and cloning procedures were carried out according to Maniatis et al. (1982). A, Apal; B, Bam Hl; Bg, BgllI;C, Clal; E, EcoRl; E47, Eco471II; H, HindllI; P, Pstl; S, SalGl; Sm, Smal; X, XhoL TABLE t Relative frequencies of recombination in Escherichia coil HBI01 recA-, HBI01 [pRECi] and C600recA ÷ strains E. colia

Relative frequencies of recombination b

HB 101recA HB 101 [pRECi ] C600recA ÷

0.0025 0.5 1.0

'~ All recipients HBl01recA -, HBI01[pRECI] and C600recA ÷ have the isogenic mutation leuB- (Maniatis et al., 1982). b GTl41euB + from the laboratory collection served as DNA donor and as Plvir host. Transduction was carried out according to Miller (1972). The relative recombination frequency was estimated in recipient strains by the number of Leu ÷ transductants. The frequency of homologous recombination in C600 recA ÷ strain was set as 1.

71 defect in E. coil recA - cells. The recA-Mfgene also partially restored resistance to UV irradiation to E. coil recAmutant (Fig. 2). The ability of the recA-Mf product to promote ~.CI repressor autocleavage was demonstrated by prophage induction. Resident prophage cannot be induced, either spontaneously or after DNA damage, in E. coli recA- cells, because this process requires an active RecA protein. DNA damage or blocked replication causes the activation of RecA-Ec for this role. Lysogenic strains of HB101(~.) [pX3], carrying the recA-Ec gene in pBR322, and HBI01(A)[pREC6], carrying the recA-Mfgene in pUCI9, were compared for spontaneous and UV-induced prophage excision (Table II). Although no phages are known for M. flagellatum, its RecA protein apparently takes part in an autodigestion of ACI repressor in vivo. Despite the amount of RecA-Mf, synthesized in HB 101(;L)[pREC6], is several times less than the amount of RecA-Ec, synthesized in HBI01(~.)[pX3] (data of SDS-PAGE, not shown), pREC6 caused more active spontaneous prophage excision than pX3 (Table ll). It might indicate that RecA-Mfprotein is more active as coprotease for the ~CI repressor than recA-Ec in the absence of DNA-damaging influences. DNA damage significantly stimulates coprotease ability of both proteins and results in increased prophage induction after UV irradiation of the lysogenic strains (Table II). (c) Nucleotide sequence of the rec,4-Mf gem The sequence of the 1349-bp fragment harbouring the recA-Mfgene and its flanking regions is shown in Fig. 3. An

~.

2- ~

[

p

U

C

l

g

l

reeA÷

"o 1O

mm

0IN gl

"|"

¢)

-2. -3

reeA". ipUCIgl

tb

zb

3'0 4b

s'0

Time of Irradiation [sl Fig. 2. UV survival. E. coil strains ofC600recA ÷ [pUCI9] (O) as well as its recA- mutant carrying pUCI9 (O); pREC6 (i.e., pUCl9::recA-Mf) (&) or pXl3 (i.e., pBR322::recA-Ec) (an) were compared for survival after UV irradiation. Strains were grown in LB to 5 x l0 s cells/ml, centrifuged, washed, and various dilutions were plated onto LB agar and irradiated with increasing dose of 254 nm radiation. 15 s corresponds to approx. 10 J/m 2. Plates were incubated at 37°C in the dark for 24 h. Surviving colonies were counted and compared to a zero-time control.

TABLE I! Spontaneous and UV-induced excision of ~. prophage Lysogenic strain HBI01(~) a carrying [plasmid] gone

[pUCI9] [pREC6] recA-Mf [pX3] recA-Ec

Yield of free phages (pfu/ml) Spontaneous b

UV induced ~ I

II

< 10 2.8 x 10s

< 10" 2.0 × 109

< 102 4.0 × 108

4.5 × 104

2.0 × 104

5.5 x i0 s

a E. coil HBi01(,~)recd-; HBI010.)[pREC6], i.e., pUCl9::recA-MJ~ HB 101(,l)[pXl3], i.e., pBR322: :recd-Ec, were used. b The cells were grown at 37°C in LB to steady state, collected by centrifugation, and supernatants were assayed for pfu number (Zaitzev et al., 1986) on C600rec,4 + lawns. c [~g-phase cultures (approx. 2 x l0 s cells/ml) were centrifuged, resuspended in M9 medium (Maniatis et ai., 1982), UV-irradiated for various times in petri plates, put into LB medium and incubated with shaking in the dark at 37°C for 1.5 h, then lysed with chloroform; cell debris was removed by centrifugation and lysates were assayed for pfu. Column I corresponds to approx. 15 j/m2; II to 30 J/m 2.

ORF from 1 to 1032 nt shows a high degree of homology with other sequenced bacterial recA ÷ genes, and the Mr of the protein is 36 933. It is in good agreement with that of plasmid pREC6-encoded RecA-Mf, observed in E. coli maxicells (Fig. 4). The G + A-rich region preceding the recA-Mfstart codon resembles an SD-like sequence (Shine and Dalgarno, 1976). The approx. 300-bp region upstream from the recA-Mf start codon has no SOS box, a conservative site for LexA repressor binding (Fig. 3). The consensus sequence of the SOS boxes of all LexA-regulated genes from enterobacteria (Little and Mount, 1982; Walker, 1984; Freudl et al., 1987) is: 5 ' - C T G T - N g - C A G - . The only substitution in this site resulted in constitutive expression of the genes in E. coil (Wertman et al., 1984). In vitro experiments (Hurstei et al., 1988) revealed the crucial role of these and adjacent bp for LexA-Ec interaction with DNA in the operator regions. The recA + genes from E. coli, Proteus mirabilis and the nonenteric bacterium, Pseudomonas aeruginosa, contain this site in their operator loci (Sano and Kageyama, 1987; Akaboshi et al., 1989) and are DNA-damage inducible. However, expression of the recA ÷ genes from several other bacteria seemed to be independent of DNA damage (Finch et al., 1986; Goodman et ai., 1987; Ramesar et al., 1989). Ramesar et al. (1989) did not find the SOS box in the 190-bp region upstream from the ORF of Thiobaciilus ferrooxidans recA + gone, but the gene was not expressed in E. coil from its own promoter.

72 -2# sggtgatgF:cctggF:F:F:tg~:ctacsF:scCF:cgtcattsgtF:ttc -240

-200

-120

-80

-ldO

~t~c~ggF:~t~F:tggF:~tgF:gg~aF:~a~cna~ccggtcggtacc~tatF:ttt~F:tgOF:cc~tcF:~at~F:c gtatcatcacccsascccaccacttccttgF:gag ,,,40

•

-1

a~a~F:c~tF:F:s~aca9t¢t9t¢~a~ac¢F:~ta~c¢9F:t~gUF:s~c~ts¢ttt~cc¢a(t9it~tst9aa~tssttF:s~F:tttaF:ag~c~act¢~saaa~tstt¢ac~g 1

40

80

ttO ~ t gas sic sos iF: ass F:O eta F:c F:c F:c ctc too cos ate tag its css ttt OF: na OF: tot sic sto oF: stg OF: Osc n~t asp glu ash art set iys its leu t i t t i t sis ltu set 91n ile 91u lys 91n'phs 91Y lys gly sortie met arO net 01y asp 1

10

ace ~,~t gtc F:t F:t thr a~p vii ala eli

•

120 9ac stc cso get gtt Oct scc OF: tcc coo OF: ctg asp ile 91n sit vii set thr 91y ser lee gly leu 40 200 90c cc9 gas tcc tcc OF: sad ace ac9 coo act tot 91Y pro 91u sir ser 91Y lys thr thr leu thr leu 70

20

160 gac arc F:c coo got stt OF: OF: ttg cc9 cgc OF: cF: asp ile Eli leu 91)' Sit gly g17 leu pro ar9 91Y art d0 5O 240

tot got ate 0c9 cat at9 cat aa9 ~09 OF: OF: act F:i sar vii tie ala tln met 91n iys leu oly 91y thr ila 9O 80 360 320 28O gtt tc¢ tat coo ctc st{ tcc coo {cc gsc F:s ttc arc tar ~ t gas Cat ~4J)¢tc gtC cog gtc tic F:O cat sag ctc got otc sat vii ser sip lee lee Sit set gin pro asp slt phe ils asp t i t flu his sis ieu asp pro vii tyr tla gln tys leu fly vii ass 110 120 100 440 400 ace OF: on cag~cg ctc gag ate F:c gee atg cog 009 cot tcc OF: tog got get 9to gto gig tic 9ac ice otc oct F:9 ct9 acg thr 91y 91u gin eli leu 91u ile ale asp net leu val art set 91Y sir vii asp val vii val vii asp set eel ale sis IH thr 140 150 130 529 400 ((( tag F:c gel ate gn OF: gn ttg OF: 01( tog cac tog 00c ctg cat gcc oF: cog ato too coo OCt ctt oF: tat ctc ace F:c pro lys ale 91u ile 91u gly 91umt 91y asp see his net 91Y leu gin ala art leu set sir tln sis lee art lys leu thr sis 180 160 170 56O 6OO sac ate at9 oF: ace tot tcc ttg gtc ate ttc tic sac cog ate oF: tog no tic ~ic ctg tog ttc 90c sac ccc gag acc acc acc ass Sit lys art thr ass thr leu vii Sit phe tie ass 01n Sis art met lys ile gly Itu art phe gly asn pro 91u thr the tit , 190 200 210 640 600 720 OF: OF: eat F:s ctc asO tic ttc F:c tcc 000 cgc ctg OaC ate cgc cF: tcc ggc gcg ttc tag tag ggo Osc 0a9 9tc sod gOt tea 01Y gly t ~ sis Isu lys phi t~ t i t sir val art leu asp ilt Jr9 art thr gly sls ile lys lys gly asp 91u vii thr 01Y u r sac 9to gag stt tic ile ca1 flu Sit tyr

220

tat uc c00 9tc 01u thr Jr9 vii

0|9 9~_.9 " tic 91u g,y 9lu Sit 00c n0 0it sic 01y lys asp tan

230

760 lug its ttg sit sic tic gtg F:c oct cog ttc is0 cot F:9 OUt ttc tic lys vtl vii lys ass nn vii tla pro pro phi lys leu sit glu phe tip 250 260 840 tic got ctc ~c gtc tic ctt , , , tO9 ,tc 0'0 '19 F:c 01t F:c 00, tic Sis 91u leu v,V vii ass Its lys lee Sit tie I n t i t 9-Y sit Set tyr 200 290 920 960 0c9 coo 019 ttc ctc 800 010 clc co0 0it ttc F:c l i t 0i0 itc tic F:c t i t art Glu phe lee set 01u hit pro glu ile i l l tat 01u i11 tip t i t 310

320

240

000 sic ctc tat OF: gas f i t ate tog cF: I1e IH t~r sly tie gly tie sir art 270 000 oF: tic |10 9|c 9'9 " 9 ,to 99c co, sir rye IVs sly 91u lys ile 91Y 9In 300 It0 tot cgc 9q cot tcc t i t ctg gcc lyJ Sit Jr| 01u his str its lsu I]t 330

1000 1040 ttc F:c tc9 eft tog to0 Des cc6 tit gn tit iF: 01c 9n t,F:cttcctccttgutF:9t0t ass t i t t18 set thr thr t i t pro tip glu 01u let asp 9|u 340

Fig 3. Nucleotidc sequence of the recA.Mfgeneand its flanking regions. The deduced primary RecA-Mfprotein sequence is given below the coding nt sequence. Restriction fragments containing the recA-Mfgene and neighbouring regions from pREC6 (Fig. 1) were subcloned in both orientations into M 13tg130 and tgl31 (Kieny et al., 1983) and sequenced by dideoxy chain-termination method (Sanger et al., 1977). Last digits of numerals are aligned with corresponding nt.

(d) Expression of the recA-Mf gene in Escherichla coil

Since the recA-Mf is cloned in pREC6 in an opposite orientation to the plasmid pUCI9 promoters (not shown), we supposed that it might be expressed from its own promoter contained in the M. flagellatum DNA fragment. The d~ta ofM. flagellatum gene expression, obtained in our laboratory, indicate that they usually can be efficiently expressed in E. coli; therefore, many M.flagellatum promoters can probably be recognized in E. coll. To investigate the recA-Mf gene expression in E. coli, recA-Mf': :' lacZo( fusions were constructed (Fig. 5) and assayed for/IGal activity. Plasmid pREC8 contains about 800 bp, pREC9 170 bp from the upstream rcgion o,~ the

recA-Mfgene and the first seven structural codons fused in frame with the lacZ~ gene. After transformation into E. coil TGI {A(pro-lac) recA + [F' traD proAB + laclq]} strain (Gibson, 1984), both plasmids conferred blue colour to the colonies on the XGai-containing LB agar without IPTG or SOS-inducing agents. It means that the putative recA-Mf promoter(s), active in E. coli, is (are) situated in both fragments. To verify this, a primer-extension experiment was carded out. Total RNA from TG I, harbouring pREC7, pREC8 or RECg, standard primer for M 13 sequencing and reverse transcriptase were used. G - 33 and C - 32 were the main recA': :' lacZ~ tsp (data not shown). Thus, in the absence of DNA-damaging agents, the recA-Mf gene is

73

a

b

SM

A ~? It.ll

lips Pz,le = pnlsc?

III

8oo

E

lacZot

bp -- agc'aaa'g~GGG~4AT'TCA

- -

67 J E ; P o l Ik + d N T P | I i 9 a s e

43 m

Pl.e~ pREC8

SM

liPS III

A ~']7 ~'1

II~JI

coo up -

/acZ~

a~Taaa'gcG'GGA'AT"T~4AT'TCA -A + ! i ; Pol |k *

i!

~., pit,c9

Fig. 4. Autoradiograph of [3SS]proteins produced by maxieells of E. coil AB2463 recA - strain (Howard-Fianders and Theriot, 1966), containing pREC6 (pUClg::recA-Mf)(lane a) and pUC 19 (lane b). M axieells were prepared according to the technique described by Hames and Higgins (1984). Soluble proteins were analysed by 0.1% SDS-10% PAGE (Laemmli, 1970) followed by autoradiography of dried gel. Marker sizes are in kDa. The 37-kDa protein is RecA-Mf; the smaller protein is a product of bla gone from pUCt9 (both are indicated by arrows).

_

dNTP; Iiguse

" .~M

^ ET~C~ I Pr~Jl lacZ~

17o bp -- ag~aaa'gc~G6A~TT'AAT'YF~ --

Fig. 5. recA-Mf: :lacZ~, fusion construction. The SalGI2-Eco47IlI fragment from pREC6 (Fig. i), carrying about 800 bp from the upstream region of the recA-Mf and several first bp from the coding part, was subcloned into pUCl9 digested with SalGI + Smal to obtain pRECT. Then pREC7 was digested with EcoRl, treated with PolIk to make blunt ends, and ligated. It allows to construct in frame fusion of several first recA-Mfcodons and the rest oflacZ~ codons, pREC9 is pREC8 shortened by the deletion of Apal-HindllI fragment distal to the recA-Mf first codon. Restriction sites which were disrupted during manipulations are included in parentheses or are shown as smF4?or ~. Abbreviations are the same as in Fig. 1. A heavy line corresponds to M.flagellatum DNA. Pl,c denotes a promoter oflacZo~ gone in pUCI9; Prec a putative promoter of recA-Mf. Direction of transcription is shown by arrows. Under each scheme of plasmid the nt sequences of the recA (lower-case letters) and lacZot (capital letters)junction are shown; each recA.Mfcodon is divided by two apostrophes; each lacZoL codon by a single apostrophe. TABLE III Levels of ~Gal activity in cultures exposed to SOS-inducing agents

transcribed in E. coil from the single promoter active region. To learn the influence of LexA-Ec repressor on the recA-Mf transcription, the strains TGI recA + carrying pREC8 or pREC9 (or pUCI9 as negative control) were exposed to the SOS inducers. Assuming that LexA-Ec represses recA.Mfexpression, the level of/~Gal in the case of recA-Mf': :' lacZ~ fusions should significantly increase after induction. However, no such induction of/~Gal was observed (Table IIl). (e) Comparison of the RecA proteins from various bacteria Inspection of the various bacterial vecA + genes revealed an obvious nt sequence similarity of the coding regions but their products appear to be much more homologous (Fig. 6). RecA-Mfprotein consists of 344 aa of which 70% are identical to the corresponding residues of RecA-Ec, 71% to RecA-Pm, 73% to RecA-Pa, and 70% to RecA-Tf protein. Taking into consideration the substitution of similar aa the level of homology is still higher. The essential differences exist in the C-terminal sequence and in the 31-39-aa region. A role for the RecA-Ec C terminus in coprotease regulation has been suggested by

E. coila TGIrecA + [plasmid]

[pUCi9] [pUCI9] + IPTG [pREC8] [pREC9]

Activity of pGal (% of control) b Control (0 h)

100(25) 100(700) 100(150) 100 (120)

10 J/m 2

30 J/m 2

MMS

lh

2h

ih

2h

Ih

2h

108 89 94 75

105 106 i!0 90

102 88 94 83

86 90 95 90

80 87 103 74

71 93 74 65

a Strains of E. coil TGlrecA ÷ A(lac.pro) [F'proAB ÷ iacl q] harbouring pUCI9 in the absence or presence of IPTG (shown as + IPTG); pREC8 t,r pREC9 were grown in M9 medium (Maniatis etal., 1982)/0.4% glucose/0.2% Casamino acids/100 pg Ap per ml to reach the late log phase, i.e., the conditions of maximal RecA-Ec inducibility (Sommer etal., 1985). b At zero time one-fourth of each culture served as eantro! or was UV-irradiated in petri plates with doses of approx. 10 or 30 J/m 2. In the fourth ease MMS (0.25%), which causes SOS response in E. coli recA ÷ cells (Casaregola et al., 1982), was added at zero time. Activity of/~Gal was measured according to Miller (1972) 0,1 and 2 h after the treatments. Values are % of corresponding ~Gal activity compared with values of untreated control, 100%, at the same time. Mean values ofpGal activity (in Miller units) for each strain at zero time are shown in parentheses.

74 Ec

Pm Nt" Pa Tt"

1 • ~ ** 60 . .t-, AIDENKQKALAAALGQ 1EKQF~GSi MRLGE_DRSMDVET 1STGSLSLDIALGAGGLRvlBR 1VEI¥ ~ T T L T L Q

M M~ RS M~ KR M.._ Q R S G S

S R S D

N M DT VAA IQAV AV M DHE QA_IPA AV DHNAIK I VY

go

V G G 6 L

i

I V

R R K R V

~;

S

H

* ILA3

150

*

Ee

V ! AAAQRE~TCAFIDAEHALDP1YARKLGVDI DN~PDTGEQALE IC'DALAlC3GAVDVIVVDSVAALTPKAEI

Pa Tt"

QMKL G A E KQA V A 5CAAGA

Ec Pet ~ Pa TF gy

I C ~ H ~ A M R K L A G N L K Q S N T L L I F I NQ1RMKIGVI~PEI'TTGGNALKFYASVRLDI RRIC~VKEGENVV V N S NOEl M Q L L TA ! RT V L T 1 K DE T M AV Q L L IT I N A C V T DE M V Q L L N TA ISR V Y I KSDE . ISY V T QTL K AKGi

Ec Pm Mr Pa

240 * * 2';'0 * * GSETRVKVVIOIKI AAWKQAEFQI LYGEGI NFYGELVDL6VKEKL.I EKAGAWYSYKGEKIGQGKANATAWLKDNPETAKE V M T I H V N NY EH MYN NV P L D SEE l i e NL D REF REH i N VSP R K YRT i l QLG V S QS AKY E IG.~;V

Pm

S

•

Tt" NO SF C1RAK A E~. /~= Mr Pa Tr

1

O

V Q D G G H

*

*

V P RE

V o

Ri

5

NVSD I NVD V LE I

A MV T MV AMV

*

AY

*

SRLS

S N

V l

210

FDIV T

O IF K SRV CML SRN QCHYARK

V

L 1 *

Q HR

D RQY VH L AN

.%52 I EKKVREI,LL.SNPN,";__T PDFSVDDSEG_VAE.,TNEDF. LNT 1, M ItAGEF SAA AGEE DSD_ DD K . DA i H _ - t.A AA~ TA ._EE: D_E. L T I DO A I L S G P V K A ...A.EEV_AD_ AD. QRI _AAAA GHPLA_ A_EEV SPQRSAS.

Fig. 6. Primary structures (in single-letter code) ofthe RecA proteins from E. coli (Sancar et al., 1980), closely related to it P. mirabilb (Akaboshi et al., 1989); as well as M.flagellatum (this work), P. aeruginosa PAO (Sano and Kageyama, 1987), T. ferrooxidans (Ramesar et al., 1989), and partially Synec~ococcus sp. strain PCC7002 (Murphy et al., 1987). The aa number are based on the RecA-Ec protein (which has no N-terminal Met). Gaps in the aa sequences (introduced to allow maximum alignment) are shown as underlines; residues identical to those orE. coli are not shown. Residues mentioned in section • are marked by asterisks. Full stops are situated after the last aa of each protein (except for RecA-$y, where points limit a sequenced part of the protein).

Ogawa and Ogawa (1986) through the analysis of truncated recA.Ec gene product properties and confirmed by analogous data for the recA.Pa gene with changed or truncated C-terminal codons (Zaitzev et al., 1986). The 31-39-aa region might take part in the control of coprotease ability by interaction with the C terminus (Sano and Kageyama, 1987). Some ofthe ReoA-Ec residues which are responsible for relevant coprotease ability in the absence of DNAdamaging agents (Wang and Tessman, 1986): 25, 38, 39, 158, 169, 179, 184, 301 aa are significantly divergent among RecA proteins. These aa substitutions may be responsible for the increased ability of pREC6 encoded RecA-Mf to promote ~CI repressor autodigestion without DNA damage. Cys 116which affects ATPase function (Kuramitsu et al., 1984) is not conserved. There is much more homology in the other parts of RecA proteins up to the identity of significant aa. For example, the Gly16°-Asp replacement was shown to disrupt all known RecA-Ec functions (Kawashima et al., 1984). All proteins have Gly at this position. A sequence hypothesized previously as adenine-binding fold, from Gly66 to Thr73 (Sano and Kageyama, 1987), is also identical. Knight et al. (1988) showed the crucial role of Tyr2~ for ATP binding by enterobacterial RecA. All but RecA.Sy have Tyr in this position. Gly =°4 and Gly 2"9, which are important for the repressor's recognition, Va1246 and lie 29s controlling thermal stability of the molecule are also conserved.

ACKNOWLEDGEMENTS

We are grateful to Dr. E. Zaitzev (B.P. Konstantinov Leningrad Institute of Nuclear Physics) for providing us with plasmid pX3, and Dr. S. Kulakauskas for C600 recA- strain. REFERENCES Akaboshi, E., Yip, M.L.R. and Howard-Flanders, P.: Nucleotide sequence of the recA gene of Proteus mirabilb. Nucleic Acids Res. 17 (1989) 4390. Better, M. and Helinskt, D.R.: Isolation and characterizatio, of the recA gene ofRkizoblum mellio~. J. Bacteriol. 155 (1983) 311-316. Casaregola, S., D'Ari, R. and Huisman, O.: Quantitative evaluation of recA gene expression in £scherlchta coll. Mol. Gen. Genet. 185 (1982) 430-438. Chistoserdov, A.Yu, Eremashvili, M.R., Mashko, S.V., Lapidus, A.L., Skvortsova, M,A., Sterkin, V.E., Strongin, A.Y. and Tsygankov, Y.D.: Expression of human interferon alfa-F 8ene in obligate methylotroph Methylobacill~ jqagellatum and Pseudomonas putida. Mol. Genet. Microbiol. Virol. (USSR) 8 (1987) 36-41. Either, G., Solonin, A.S. and Tanyashin, V.I.: Cloning ofa recA-like gene of Proteus mirabilb. Gene 14 (1981) 301-305. Finch, P.W., Brough, C.L. and Emmerson, P.T.: Molecular cloning of a recA-like 8ene ofMethylophgus methylotrophus and identification of its product. Gene 44 (1986) 4753--4"/62. Foster, P.L. and Sullivan, A.D.: Interaction between epsilon, the proofreading subunit of DNA polymerase I!!, and proteins involved in the SOS response of F.scht~hla coll. Mol. Gen, GeneL 214 (1988) 467-473.

75 Freudl, R., Braun, G., Honort, N. and Cole, S.T.: Evolution of the enterobacterial sum gene: a component of the SOS system encoding an inhibitor of cell division. Gene 52 (1987) 31-40. Gibson, T.J.: Studies on the Epstein-Barr Virus Genome. Ph.D. Thesis. University of Cambridge, Cambridge, England, 1984. Goldberg, I. and Mekalanos, J.J.: Cloning of the Vibrio cholerae recA gene and construction of a W~briocholerae recA mutant. J. Bacteriol. 165 (1986) 715-722. Goodman, H.J.K., Parker, J.R., Southern, J.A. and Woods, S.R.: Cloning and expression in EschericMa coli of a recA-iikegene from Bacteroides fragilis. Gene 58 (1987) 265-271. Hames, B.D. and Higgins, SJ.: Transcription and Translation. A Practical Approach. IRL Press, Oxford. 1984. Hamood, A.N., Pettis, G.S., Parker, C.D. and Mclntosh, M.A.: Isolation and characterization ofthe Vibrio cholerae recA gene. J. Bacteriol. 167 (1986) 375-378. Horii, T., Ogawa, T. and Ogawa, H.: Organization of the recA gene of Escherichia coli. Proc. Natl. Acad. Sci. USA 77 (1980) 313-317. Howard-Flanders, P. and Theriot, L.: Mutants of EschericMa colt K-12 defective in DNA repair and genetic recombination. Genetics 53 (1966) 1137-1150. Hurstel, S., Granger-Schnm'r, M. and Schnarr, M.: Contacts between the LexA repressor - or its DNA-binding domain - and the backbone of the recA operator DNA. EMBO J. 7 (1988) 269-275. Kawashima, H., Horii, T., Ogawa, T. and Ogawa, H.: Functional domains of£sche~chia coli recA protein deduced from the mutational sites in the gene. Mol. Gen. Genet. 193 (1984) 288-292. Keener, S.L., McNamee, K.P. and McEntee, K.: Cloning and characterization of the recA genes from Proteus vulgags, Erwinla carotovora, Sh~ella fiexneg and Escherichia coli B/r. J. Bacteriol. 160 (1984) 153-160. Kieny, M.P,, Lathe, R. and Lecocq, J.-P.: New versatile cloning and sequencing vectors based on bacteriophage MI3. Gene 26 (1983) 91-99. Kletsova, L.V., Govorukhina, N.I., Tsygunkov, Y,D. and Trotsenko, Y.A,: Obligate methylotroph Methyiobactl!us flagellatum KT metabolism. Microbiologia (USSR) 56 (1987) 901-906. Knight, K.L., Hess, R,M, and McEntee, K.: Conservation of an ATPbinding domain among RecA proteins from Proteus vulgarls, Erwinta carotovora, Sh~ella flexnert, aod Escherlchta colt K-12 and B/r. J. Bacteriol. 170 (1988) 2427-2432. Kokjohn, T.A. and Miller, R.V.: Molecular cloning and characterization of the recA gene of Pseudomonas aerufinosa PAO. J. Bacteriol. 163 (1985) 568-572. Koomey, J.M. and Falkow, S.: Cloning of the recA gene of Neisseria gonorrhoeae and construction of gono¢:occal recA mutants. J. Bacteriol. 169 (1987) 790-795. Kuramitsu, S., Hamaguchi, K., Tachibaba, H., Horii, T., Ogawa, T. and Oguwa, H.: Cystenil residues of Eschegchta coli RecA protein. Biochemistry 23 (1984) 2363-2367. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Lanzov, V.A.: Bacterial RecA protein: biochemical, genetic and physicochemical analyses. Genetika (USSR)21 (1985) 1413-1427. Little, J.W.: Antodigestion of LexA and phage repressors. Proc. Natl. Acad. Sci. USA 81 (1984) 1375-1379. Little, J.W. and Mount, D.W.: The SOS regulatory system of£scherichia coll. Cell 29 (1982) 11-22. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Miller, J.H.: Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972. Murphy, R°A.,Bryant, D.A., Porter, R.D. and de Marsac, N.T.: Molecular

cloning and characterization of the recA gene from cyanobacterium S vnechoceccus sp. strain PCC7002. J. Bacterioi. 169 (1987) 2739-2747. Nohmi, T., Battista, J.R., Dodson, L.A. and Walker, G.C.: RecAmediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation. Proc. Natl. Acad. Sci. USA 85 (1988) 1816-1820. Ogawa, H. and Ogawa, T.: General recombination: functions and structure ofrecA protein. Adv. Biophys. 21 (1986) 135-148. Ohman, D.E., West, M.A., Fiynn, J.L. and Goldberg, J.B.: Method for gene replacement in Pseudomonas aeruginosa used in construction of recA mutants: recA-independentinstability of alginate production. J. Bacteriol. 162 (1985) 1068-1074. Paul, K., Grosh, S.K. and Das, J.: Cloning and expression in £. coli of a ratA-like gene from Vibrio cholerae. Mol. Gen. Genet. 203 (1986) 58-63. Ramesar, R.S., Woods, D.R. and Rawlings, D.E.: Cloning and expression in Escherichia coli of a recA-likegene from the acidophilic autotroph Thiobacillus ferrooxidans. J. Gen. Microbiol. 134 (1988) 1141-1146. Ramesar, R.S., Abratt, V., Woods, D.R. and Rawlings, D.E.: Nucleotide sequence and expression of cloned ThiobaciUusferooxidans rec.4 8ene in Escherichia coil Gene 78 (1989) I-8. Resnick, D. and Nelson, D.R.: Cloning and characterization of the Aeromonas caviae recA gene and construction of an A. caviae recA mutant. J. Bacteriol. 170 (1988) 48-55. Sancar, A., Stachelek, C., Konigsberg, W. and Rupp, W.D.: Sequences ofthe recA gene and protein. Prec. Natl. Acad. Sci. USA 77 (1980) 2611-2615. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Sano, Y. and Kageyama, M.: The sequence and function of the recA gene and protein in Pseudomonas aerufinosa PAO. Mol. Gen. Genet. 208 (1987) 412-419. Shine, J. and Dalgarno, L.: Determinant of cistron specificityin bacterial ribosomes. Nature 254 (1976) 34-38. Slilaty, S.N. and Little, J.W.: Lysi~e-156 and serine-! 19 required for LexA repressor cleavage: a possible mechanism. Pr~lc. Natl. Acad. Set. USA 84 (1987) 3987-3991. Sommer, S., Ballone, A. and Devoret, R.: SOS induction by thermosensitire replication mutants of mini-F plasmid. Mol. Gen. Genet. 198 (1985) 456-464. Vericat, J.-A., Guerrero, R. and Barbe, J.: Increase in plasmid transformation efficiencyin SOS-induced £scherichta coli cells. Mol. Gen. Genet. 21 ! (1988) 526-530. Walker, G.A.: Mutagenesis and inducible response to deoxyribonucleic acid damage in Escherichta colt. Microbiul. Ray. 48 (1984) 60-93. Wang, W.-B. and Tessman, E.S.: Location of functional regions of the Escherichta colt RecA protein by DNA sequence analysis of RecA protease.constitutive mutants. J. l~acteriol. 168 (1986) 901-910. Wertman, K.F., Little, J.W. and Mount, D.W.: Rapid mutational analysis of regulatory loci in £scherichta colt K-12 using bacteriophage MI3. Proc. Natl. Acad. Sci. USA 81 (1984) 3801-3805. Yanisch-Perron, C., Vieira, J. and Messing, $.: Improved MI3 phage cloning vectors and host strains: nucleotide sequences of the M 13rap18 and pUCI9 vectors. Gene 33 (1985) 103-119. Yankovsky, N.K., Fonstein, M.Y., Lashina, S.Y., Bukanov, N.O., Yakubovich, N.V., Ermakova, L.M., Rebentish, B.A., janulaitis, A.A. and Debabov, V.G.: Phasmids as effective and simple tools for construction and analysis of gene libraries. Gene 81 (1989) 203-210. Zaitzev, E.N., Zaitzeva, E.M., Bakhlanova, i.V., Gorelov, V.N., Kuzmin, N.P., Kryukov, V.M. and Lanzov, V.A.: Cloning and characterization of recA gene from Pseudomonas aeruginosa. Genetika (USSR) 22 (1986) 2721-2727.