Identification of the integration host factor genes of Erwinia chrysanthemi 3937

Biochimie (1994) 76, 1055--1062 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

1055

Identification of the integration host factor genes of Erwinia chrysanthemi 3937* A Douilli6, A Toussaint, M Faelen** Laboratoire de G~n~tique. Unit~ Transposition bact~rienne, UniversitE Libre de Brltrelles, 67. rue des Chevmtr, B-1640 Rhode-Saint-Gen~se, Belgium

Summary - - Two Erwinia chrysanthemi homologues of the hinL4 and hired genes of Escherichia co!i which encode the integration host _factor (IHF) were cloned, sequenced and compared to their homolog in other enterobacteria (EMBL accession nos X74749 and X74750). Both genes were inactivated by the insertion of an antibiotic resislance cassette, allowing for the isolation of IHF- mutants of E chr3,santhemi. IHF I himA I hired I E chrysanthemi

Introduction

Materials and methods

T h e i_ntegration host _factor (IHF) o f Escherichia coli is a small basic heterodimeric protein c o m p o s e d o f subunits encoded by the himA and hireD (or hip) genes (for a review see [ I 1). "Ibis 'histonelike' protein binds to a consensus recognition sequence on D N A ( W A T C A A N N N N T T R ; [21) and introduces sharp bends o f about 140 ° in the double helix 13--61. It is k n o w n to be required for several types o f site-specific recombination reactions, to participate in the initiation o f the replication o f plasmids and to be involved in the regulation o f different g e n e s o f E coli and o f s o m e o f its bacteriophages [ I, 71. A l t h o u g h dispensable, I H F is widely distributed a m o n g eubacteria and has been detected in E coli, Salmonella o,phimurium, Serratia marrescens, Rhodobacter capsulatus, Haemophilus influenzae and Aeromonas proteolytica [8-11 ]. Erwinia ehrysanthemi, a phytopathogenic enterobacterium responsible for soft rot diseases must also contain an I H F h o m o l o g u e since it allows the lytic growth of p h a g e Mu, which requires I H F [ 12, 13]. In order to further evaluate the distribution o f IHF as well as the conservation o f gene organization in the n e i g h b o r h o o d o f the himA and himD genes, we characterized the him genes o f the E chrysanthemi 3937 strain [ 14].

Bacterial sn'ains, phages and plasmids

*This paper is dedicated to Ariane Douilli6, who passed away after a painful disease early 1992. **Correspondence and reprints.

Bacteria, phages and plasmids are listed in table I.

Media Strains were grown in LB medium [28] and titrated on LA plates [28] containing 1.2% Difco agar. Minimal medium was 132 [29] supplemented with I% glucose and contained 1.4% Difco agar. Kanamycin (Km, 10 lag/ml), chloramphenicoi (Cm, 12.5 [ag/mi), ampicillin (Ap, 10 lag/ml for the selection of MupAp5 lysogens, 50 lag/ml for selection of plasmids), tetracyclin (Tc, 10 pg/ml), spectinomycin (Sp, 50 pg/ml) and streptomycin (Sin, 25 lag/ml) were included when appropriate.

Methods for phage manipulations The general methods used in the manipulation of phage have been previously described [30, 31]. Lysogenization by Mucts 62pAp5 or Mucts62pKm7701"G +'' was assayed by spotting 10 lal of serial dilutions of lysates on lawns of the strains to be tested on LA plates supplemented with Ap or Km. The titre of the lysates was determined on a lawn of 3937 on LA plates. The plates were incubated overnight at 30°C. The frequency of lysogenization was calculated as the ratio between the title of Ap R or Km R colonies and the title of the lysates on 3937 [32].

Screening of the E chrysanthemi genomic library for the rpsA gene A 3937 genomic library was constructed by in vitro packaging of BamHl linearized pRK7813 cosmid, ligated to :t: 25 kb long fragments obtained upon partial digestion of 3937 total DNA with Sau3A [33]. ~c!857Sam7 lysates were prepared on

1056 Table I. Bacterial strains, plasmids and phages. Genotype symbols are according to Bachmann [ 15].

Genotype/characwristics Bacwria E chrysanthemi 3937

E coli EJ J320 NP37 N99 himA42 MC252 MB3001(~ .÷) G J23 Sl7-1

Reference~source

Wild type strain

[ 14]

thrS1029 argE3 lacYl galK2 manA4 mti-I supE44 rpsL700 tsx-29 fhuA22 garBlO ompF627 pheSS fadL701 relAI pit-lO spoTi phoM510 gall( lacZ rpsL himA42

[161

A(lac, pro )xHiargEam gyrA rpoB supF ~A3himD::CmR leu.,, trp~., lacZ~mgall(.., galE tsx relA sueA sueB sueC sueC supD43.74 rpsA~, rpsL J~z'+ JC2926 carrying plasmids R64drdl I and pGJ28 pro. recA, hsdR with a chromosomally integrated

[171

[181 M Chandler collection

[191 [201 [211

RP4-2-Tc::Mu-Km::Tn7

Plasmids pO572

[221

pBR325 with a 9 lib long Pstl fragment of the E cht?,santhemi 3937 chromosome

This work

pULBI30

pO572 with a f~ cassette in the Smal site of himA

pO467

pBR325 with a 6.5 kb long Hindill t'ragment of the E chry:;anthemi 3937 chromosome

pTZI8R

Cloning vector deriving from pUC 18

pRK7813

Mini-RP4 cloning vector containing a Tc R marker and the kcos site

[221 1231 1241 1251

pCL 192 I

Mini-pSC 10 ! cloning vector containing a SpR/Sms cassette

pULBI31

pCLI921 with a 4 kb long Pstl fragment of E chrysanthemi DNA including the himD gene

This work

pULB 132

pULB 131 with a Cm R cassette in the Sa¢,ll site of hireD

This work

Phages Mucts62pAp5

Mucts62 with a substitution of the 6. I kb to 7.2 kb region from the c-end by a 1.1 kb fragment of Tnl

N Symonds collection

Derivative of Muc'ts62pfKm770 ! with intact G and [~ regions

Laboratory collection

Mucts62pKm 7701,,G +,,

Laboratory collection

~'!857Sam7 D108

[261

~80

Laboratory collection

OEC i. ¢bEC2 and OEC4

1271

E c'hrysanthemi specific phages

cultures of pooled clones of the library to package the TcR cosraids. The lysates were used to infect E c'~di MB3001(~.c +) which does not grow at 42°C due to the combination of a rpsAam and a supDts mutation [191. Since 3937 does not contain any sup mutation, selection of TcR transductants at 42°C was expected to provide only cosmids carrying a fun,:tionai rpsA gene.

Conjugal u'ansfer of plasmids Cultures of 3937 and of E ('off carrying the mobilizable and the adequate helper plasmid were grown overnight to saturation. Ten pi of both cultures were mixed on LA and the plates were incubated at 30°C overnight. The bacteria were resuspended in I ml of 10 mM MgSO4 and streaked at 30°C on appropriate

!057 selective minimal media to select for 3937 exconjugants containing the mobilizable plasmid.

DNA manipulations Plasmid DNA was routinely isolated by the alkaline lysis procedure [34]. Restriction enzymes and 1"4 DNA ligase were purchased from Pharmacia or Boehringer and used as recommended. Restriction fragments were analyzed on horizontal slab gel as described by Maniatis et a11351. DNA fragments for cloning were purified from agarose gels using a Gene Clean Kit (Stratagene). Transformation was carried out as described by Maniatis et al [351.

A 3.8 kb long Nrul fragmentof the 3937 DNA carded by pO572 was subcloned in the Smal site of p T Z I 8 R 123]. The resulting plasmid again complemented the E coil himA strain; it was used to sequence the 3937 himA gene, using the dideoxy-nucleotide method [41l. As shown in table II, the E chrysanthemi and E coil himA genes have the same size (ie 300 bp [32]) arid the two genes display 81% identity. At the amino-acid level, the two HimA polypeptides differed by only four out of 99 amino-acids and three of these

Construction of the CnPe cassette Ligation of the 1815 bp Scal/Psd restriction fragment of pBluescript SK+ to the 1143 bp Scal/Pstl restriction fragment of pBluescript KS+ led to the formation of a recombinant plasmid with a palindromic multiple cloning site: Sacl-Sacll-

II

II

I

Xmalll-Xbal-Spel-BamHI-Smal-Pstl-Smal-BamHI-Spel-XbalXmalll-Sacll-Sacl, The 1,8 kb long Pstl restriction fragment of Tn9 containing the cat gene was inserted in the Pstl site of the palindromic multiple cloning site, providing a CmR cassette that can be excised by a variety of restriction endonucleases including Sacil.

p0572

Sequence comparison The gap program of the UWGCG software 1361 was used to

determine sequence identity and/or similarity.

j||l

Results and discussion

j i

Identification and sequencing of the E chrysanthemi himA gene While sequencing a 1.9 kb fragment of the E chrysanthemi 3937 chromosome containing the kdgR regulatory gene, Reverchon et al [22] found that a 46 base pair region located downstream the 3937 kdgR gene showed strong homology to the promoter of the E coil threonyl-tRNA synthetase (thrS) gene. On the E coli chromosome, thrS is located upstream of a region including the inJU, rpIT, pheS, pheT, himA and btu genes I37--40]. Two pBR325 derivatives, which carried partially overlapping restriction fragments originating from the 3937 kdgR region I221, were tested for the presence of the thrS, pheS and himA genes by complementation of E coil strains mutated in the corresponding genes, pO467, which contained a 6.5 kb long Hindlll fragment, complemented thrS and pheS mutations while pO572, which carded a 9 kb long PstI f'agment (see fige 1), complemented pheS and himA mutations (as tested by the ability to plate phage Mu and ~ 8 0 [12, 13]). This indicated that each of these plasmids contained the equivalent of a pair of the three genes tested and that the relative order of these genes on the E chrysanthemi and E coil chromosomes was likely to be the same (ie thrS-pheS-himA).

pULB131 ~"

hireD v

I1 11

I

•

,

1

m_.

Fig 1. Structure of pO572 and pULB 131. pO572 carries the E chrysanthemihimA gene and corresponds to a 4.5 kb Pstl restriction fragment of 3937 inserted in the Pstl site of pBR325 [221. pULBI31 contains the himD gene and was obtained by cloning a 4 kb Pstl restriction fragment of 3937 in the Pstl site of the multiple cloning site of pCLI921. The E chrysanthemi DNA is boxed. Only the restriction sites used in this work are represented.

1058

Table lq. Identity and similarity between homologous genes and polypeptides of E chrysanthemi and E coil. Comparison of the amino-acids and nucleotides sequence was made using the 'Gap' program of GCG. Locus

Genes

Polypeptides

E chrysanthemi

E coil

Size~

Identity

Sizea

Similarity

Identity

hiwA hireD

himA himD

300 285

81.00 80.00

99 94

98.99 94.68

95.96 9 ! .49

hemC c

hemC

267 b

77.53

89 b

94.38

84.27

pheT cyac arbB d cyaFc arbFd arbG d aroL •

pheT cya bglB cyaF bglF bglG aroL

582 b

77.15 75.59 68.67 66.67 60.76 60.77 55.75

193b 848 464 106 622 276 173

90. ! 6 90.08 82.54 78.30 74.35 73.65 69.36

80.3 82.88 7 ! .55 67.93 54.81 53.79 5 ! .45

2547 1395 321 1878 828 522

aThe number of overlapping nucleotides (or amino-acids) between the two homologous genes (or polypeptides), bDue to the incomplete sequencing of the corresponding gene in E chrysanthemi, these comparisons only concern the ultimate nucleotides (amino-acids) of the carboxyl end nf the homologous genes (polypeptides). c'd.eData obtained with the E chrysanthemi B374, 3665 and NCPPB 1066 strains respectively. changes were conservative (fig 2). The two carboxyterminal amino-acids were different, the last one corresponding to the non-conservative substitution. Comparison of four HimA proteins (E chrysanthemi, E coil, S marcescens and S typhimurium) suggests that the ultimate C-terminal amino-acid is dispensable and that the nature of the penultimate amino-acid is not important for the protein function. The 582 bp region located upstream from the 3937 himA gene shows strong similarity to the end of the

E coli p h e T gene (table II). This, in conjunction with complementation data obtained with pO467 and pO572 plasmids, indicates that the gene order on the 3937 chromosome is pheS, pheT, himA as in E coil. Mechulam et al [43] have characterized the multiple promoters of the E coil himA gene. One of the promoters, p4, was localized within the p h e T gene; it contains, between the - 3 5 and - I 0 regions, an IHF binding site that is involved in the autoregulation of himA. The pheT gene of E chrysanthemi contains

HimA E E S S

chrysanthemi coli marcescens typhimurium

MALTKAEMSEYLFEKLGLSKRDAKELVELFFEEVRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITARRVVTFR•GQKLKSRVENAS•KE• MALTKAEMSEYLFDKLGLSKRDAKELVELFFEEIRRALENGEQVKLSGFGNFDLRD~`~QRPGRNPKTGEDIPITARRVVTFR~GQKLKSRVENASPKDE MALTKAEMSEHLFEKLGLSKRDAKDLVELFFEEVRRALENGEQVKLSGFGNFDLRDKNQRPGRNPKTGEDIPITAR~VVTFR~GQKLKSR~NASPKGMALTKAEMSEYLFDKLGLSKRDAKELVELFFEEIRRALENGEQVKL•GFGNFGLRDKNQRPGRNPKTGEDIPITARRVVTFR•GQKLK•RVESASPKEE

HireD E chxysdnthemi E co2i S marcescens

MTKSELIERLAGQQSHIPAKVVEDAVKEMLEQMATTLASGDRIEIRG•G•F•LHYRAPRVGRNPKTGEKVELEGKYVPHFKPGKELRDRANIYG MTKSELIERLATQQSHIPAKTVEDAVKEMLEHMASTLAQGERIEIRGFGSFSLHYRA~RTGRN~KTGDKVELEGKYVPHFKPGKELRDRANI~G MTKS ELI ERLAGQQS HI PAKAVED~KEMLEHMAATLAEGERI EI RGFGSFSLHYRAPRVGRNPKTGDKVELDGKYVPHFKPGKELRDRANI yG

Fig 2. Comparison of the amino-acid composition of the two IHF subunits of different enterobacteria. The amino-acid sequences of the E coil HimD and of both S marcescens IHF subunits are from Haluzi et al [8]; the sequences of the E coli and S typhimurium HimA polypeptides are from Miller [42] and Li el al [I0] respectively. (*), match across all sequences; (.), conservative substitutions.

1059 E e.htTmaaelma= E¢~2/

371 5127

AGTGCAAGA~GTTGGCGC~TCAGTTA~TTGGCGTA~CTTATTTGAC I II IIIIIIIIIIIII IIIIIII IIIIii11111111111111111 AATGTAAGAAAb~I"fGGCGTAAATCAGGTAGTTGGCGTAAACTTATTTGAC ...plmf [-3S] ********* [-10]

421

5177

IIIIIIII II IIIII II I I I I I IIIIIIIIIII II I I II II I I I I II GTGTACCGCGGTAAGGGTGTTGCGGAGGGGTATAAGAGCCTCGCCATAAGCCTGATCCTG

481

CAG,CC;CTC',ACAGC,C C, ;C, ,TGCC;C,ACCGT,GCGC , CGT,

5237

II i l l l l l IIIIIIII I IIIIIIIII IIIIIIIIIIIIII II Illi III CAAGATACCAGCCGTACACTCGAAGAAGAGGAGATTGCCGCTACCGTCGCCAAATGTGTA

541 52:)"/

GCAGCACTAAAACAGCGATTCCAAGCATCCTTGAGGGATT. A A C C T ~ C G C T T A C T A A I Ill Illll Illlllllll IIIII IIIIIIIlll IIIIIIIIIlllllll II GAGGCATTA~AAGAGCGATTCCAGGCATCATTGAGGGATT(E%ACCT&~GGCGCTTACAAA SD

Fig 3. Comparison of the promoter region of the himA gene of E chrysanthemi and E coil. The sequence of the E coil himA gene is from Miller [42]• The IHF binding site located between the -35 and -I0 regions of the E coil I)4 promoter of himA [43] is indicated (***). The Sh~ne-Dalgamo sequences (SD) are underlined; bold and bold italicized characters represent stop and start codons respectively. identical --35 and - i 0 regions which are separated by a DNA sequence that differs by one nucleotide from the IHF binding site identified in the equivalent E coil DNA fragment (fig 3). This suggests that the synthesis of the E chrysanthemi HimA product is also regulated by IHF. Identification and sequencing of the E chrysanthemi himD gene From the above results, it was reasonable to suspect that, in 3937, a close linkage could exist between hireD and rpsA genes which in E coil are only 160 bp apart [44]. A 3937 genome library constructed in the pRK7813 TcR cosmid [33] was screened for a plasmid able to complement the E coil rpsA strain (see Materials and methods). One such plasmid was identified and shown to complement a hireD mutation as well; when transduced in MC252 (an E coil hired mutant), it restored the ability to plate Mu and ~80. pCL 1921, a mini-derivative of pSCI01 [25] which is known to require IHF for its replication [45], was used to subclone a 4 kb Pstl fragment of the cosmid containing the hireD gene. The insert of the resulting plasmid (pULBI31; see fig 1) allowed for its maintenance in an E coil hireD and conferred to that strain the ability to support Mu and (1)80 growth. This plasmid did not complement the E coli rpsA mutation. A 400 bp long PvuI/HindIII restriction fragment of pULBI31 was purified and ligated to a pTZ18R vector digested with HindlI and HindIII. This new insert contained an intact himD gene since it comple-

mented the hireD deficiency of MC252. It was used to sequence the gene which has the same size (285 bp; table II) as its E coil equivalent [44]. Comparison between the amino-acid composition of the E coli K 12 and E chrysanthemi 3937 HireD polypeptides (fig 2) shows that the two proteins only diverge by eight out of 94 amino-residues and that all the substitutions are conservative. Interestingly, five of these differences occur at one of the six positions where a substitution had already been found between the HimD product of E coli and S marcescens [8]. Isolation of himA and himD mutants orE chrysanthemi The pO572 plasmid carries a unique SmaI site located within the E chrysanthemi himA gene. An omega cassette with SmaI ends conferring resistance to spectinomycin and streptomycin [46] was introduced in that Smal site. The plasmid obtained (pULB 130) was unable to complement the E coli himA mutant. The pULB 130 plasmid was introduced by transformation into E coil G J23 which has the ability to mobilize pBR322 derived plasmids [20]. One of the transformants was used to transfer pULB 130 into 3937. A 3937 that spontaneously lost its plasmid and acquired the himA::f~ mutation in the chromosome by homologous recombination was isolated. The pULB131 plasmid contains an unique SacIl site located within the E chrysanthemi hireD gene. Introduction of a Cm R cassette (see MaterJals and methods) with SaclI ends at that site provided a plas-

1060 reported here indicate that E chrysanthemi IHF mutants grow more slowly that their IHF+ parent in LB at 30°C. When cultured in the same conditions, the E coli IHF- and IHF + strains have similar generation times (+_ 42 min). The absence of IHF seems thus more detrimental for E chrysanthemi than it is for E coli.lt could be that in E chrysanthemi, some essential gene(s) require(s) IHF to be efficiently expressed. Alternatively, another histone-like protein could partially compensate the IHF deficiency and this function could either only exist in E coli or be more efficient in E coli than in E chrysanthemi.

mid with a himD::Cm a mutation (pULBI32). The himD::Cm a mutation was introduced in the pRK7813 cosmid containing the hireD gene by recombination. SI7-1, an E coli strain able to mobilize .pRK7813 [21], was transformed with the himD::Cm ~ cosmid. The resulting strain was used to uansfer the mutated cosmid to 3937 and the mutation was introduced into lhe chromosome by recombination.

Properties of the E chrysanthem himA and hireD genes Comparison of the E chrysanthemi himA and hireD mutants with their parental strain indicated that the him mutations had multiple effects. Indeed, the absence of either HimA or HireD: i) decreased the growth rate of the strain; generation times of 45, 75 and 80 rain were measured in LB at 30QC for 3937 and the himA and himD mutants respectively; ii) reduced the efficiency of plating of all the E chrysanthemi phages tested; OECI, OEC2, OEC4, DI08 and Mu did not form plaques on either the himA or the himD mutant; iii) prevented the production of phage particles upon induction of a Mucts62pAp5 iysogen; and iv) decreased by a factor of 10 the frequency of lysogenisation by both Mucts62pAp5 and Mucts62p Km7701"G ÷'' phages. This last result differed from what was observed with E coil where Mu lysogenisation does not appear to be noticeably affected by the himA and hireD mutations. Preliminary results also indicated that the himA::~ mutation reduces the pathogenicity of 3937 and changes the pattern of pectate lyase synthesis of the strain (F van Gijsegem, unpublished results). IHF is not essential for the survival of E chrysanthemi and E coil in complete medium. The results

Conse.~.'atio~ of the himA and hireD genes The HimA and HimD subunits of E coil K12 and E chrysanthemi 3937 seem to be particularly well conserved (95.96 and 91.49% identity respectively). As indicated in table If, this similarity is higher than that previously obse~ed between the art~3, F and B genes of E chrysanthemi 3665 and the equivalent E coli K 12 bglG, F and B genes (53.79, 54.81 and 71.55 identity respectively [47,48]), between the Cya and CyaF proteins of E chrysanthemi B374 and E coil K I2 (82.88 and 67.93 identity respectively [49]) and between the shikimate kinase of E chrysanthemi NCPPB 1066 and E coil KI2 (51.45% identity [50, 51]). These are the only 'complete' equivalent sequences available for E chrysanthemi and E coli in the EMBL sequence data bank. However, this comparison can be further extended by taking into account two additional couples of genes, hemC and pheT, that were partially sequenced in E chrysanthemi st~ins B374 and 3937 respectively: for hemC, 84.27 identity is observed between the last 89 amino acids of the carboxyi end of

Table IlL Divergence of different E coil and E chrysanthemi equivalent genes compared at first, second and third codon positions. The divergence between the three positions in the different codons of the E coli and E chrysanthemi equivalent sequences is expressed as the percent of non-identical bases and was determined using the 'Pretty' program of the 'GCG" Software.

Locus

Codon position

E coli

E chrysanthemi

First

Second

Third

himA himD hemC pheT cya bglB cyaF bgiF bglG aroL

himA himD hemC pheT cya arbB cyaF arbF arbG m~L

2.33 2.10 5.49 5.49 5.31 7.96 8.41 11.03 12.39 13.28

0.66 8.07 1.75 3.09 2.72 4.62 7.78 7.96 6.96 9.65

16 9.82 15.35 14.26 16.45 18.74 17.13 20.26 19,88 21.31

1061 the corresponding polypeptides [49] while for pheT, 80.3 identity is detected between the 193 C-termina! amino acids of the two proteins. In addition, it shoulo be mentioned ~at comparison of the different E coli and E chrysanthemi equivalem genes at fi:,,~, second and third positions indicates a better conservation of the bases located at first position for both of the two IHF subunits (see table III). The high similarity in IHF encoding genes and their tight linkage with the same genes in E coli, E chrysanthemi and S marcescens could indicate that this set of genes corresponds to a cassette that has been quite recently acquired by all these bacteria. Another possible interpretation of these observations is that the interaction between the two subunits required for IHF activity forced their conservation and that the preservation of the tight linkage with the same genes results from some unknown selective pressure that kept IHF genes next to genes involved in protein synthesis. Miller [42] noticed a strong similarity between the two E coil IHF subunits and HUtx and HUll, the two subunits of the HU historic-like heterodimeric protein. Drlica and Rouvi~:re-Yaniv [52] extended the comparison to equivalent proteins from other bacteria which revealed two highly conserved regions. As expected, none of the amino-acid differences observed between the HimA and HireD products of E coil K 12, E chrysanthemi 3937, S marcescens and S typhimurium is in these regions. We are in the process of characterizing 3037 hupA and hupB genes which should show whether mutations in the HUtz and HU[$ encoding genes also display different phenotypes in the two bacterial species. Acknowledgments We are very grateful to Dr S Reverchon for helpful suggestions and for providing some of her plasmids, to Drs BJ Bachmann and K Isono for sending some of the E coli mutant strains, to Dr G Bonu for teaching us some of the subtilities of the 'GCG' Software, toDr F Van Gijsegem for her E cho,santhemi DNA library and to Dr G Maenhaut-Michel for reading the manuscript. This work was supported through a grant from the Fonds National de la Recherche $cientifique (Credit aux Chercheurs 1.5.041.91F), AT is Mai'tre de Recherche from the 'Fonds National de ia Recherche Scientifique' (Belgium). References I Friedman D1.(1988) Integration host factor: a protein for all reasons. Cell 55, 545-554 2 Goodrich JA, Schwartz ML, McClure WR (1990) Searching for and predicting the activity of sites for DNA bioding proteins: compilation aod analysis of the binding sites for Escherichia ('oH integration host fac:or (IHF). Nucleic Acids ICes 18, 4993-5000 3 Prentki P, Chapdler M, Galas DJ (1987) Escherichia coil integration host factor bends the DNA at the ends of IS/and in an insertion hotspot with multiple IHF binding sites. EMBO J 6. 2479-2487

4 Robertson CA. Nash HA (1988) Bending of the bacteriophage iambda attachment site by Escherichia cob imcgtation host factor. J Biol Chem 263, 3554-3557 5 Stenzel "IT. P'atel P, Bastia D (1987) The integration host factor of Escherichin coli binds to bent DNA at the origin of replication of plasmid pSCI01. Cell 49. 709-717

6 Thompson JE Landy A (1988) Empirical estimation of protein-induced DNA bending angles: application to lambda site-specific mmmbination complexes. Nucleic Acids Res 16. 9687-9705 7 Freundlich M, Ramani N. Mathew E, Sirko A, Tsui P (1992) The role of integration host factor in gene expression in Escherichia coli. Mol Microbiol 6. 2557-2563 8 Haluzi I-I, Goitein D. Koby S. Mendeison l, Teff D, Mengeritsky G. Giladi H, Oppenheim AB 0991) Genes coding for integration host factors are comerved in gram-negative bacteria. J Bacwrioi 173, 6297-6299 9 Hw~ng ES, Scocca JJ (1990) Interaction of integration host factor from Escherichia ('off with the integration region of the Haemophilus il~uen-oe bacteriophage HPl. d Bacwriol 172, 4852--4860 IO Li ZJ, Hillyard D, Higgins P (1989) Nucleotide sequence of the Salmonella ryphimurium himA gene. Nucleic Acids Res 17, 8880 11 Toussaint B, Bose C, Richaud P, Colbeau A, Vignais PM (1991) A mutation in a Rhodobacwr capsulatus gene encoding an integration host factor-like protein impairs in vivo hydrogena.~e expression. Pro(. Natt Acad Sci USA 88, 10749-10753 12 Miller Hi, Kikuchi A, Nash HA, WeL,berg RA, Friedman D! (1979) Site-specific recombination of bacteriophage ~.: the role of host gene products. Cold Spring Hm'bor Syrup Quam Bio! 43, ! 121-1126 13 Yoshida RK (1984) InhibltMn of phage Mu development in E coil hinM mutams. PhD thesis, University of Wisconsin. Madison 14 Kotoujansky A, Lemattre M, Boistard P (1982) ! Itilization of themmsensitive episome bearing transposon TnlO to isolate Hfr donor strains of glwi,ia ('amtm'ora subsp chrysandwmi. J Ba('wriol I,'i0, ! 22-13 I 15 Bachmann BJ (199{l) Linkage map of E,v('herichia coil K-12, Edition 8. Miclwbiol Rev 54, 130-197 16 Johnson EJ, Cohen GN, Sant-Gin .s i (1977) Threonyl-mmsfer ribonucleic acid synthetase and the regulation) of th," threonine operon in Escherichia coil. J Bacteriol 129, 66-70 17 Eidlic L, Neidhart FC (1965) Protein and nucleic acid synthesis in two mutants of Escherichia t',fi with temperature-sensitive aminoacyl ribonucleic acid synthetases. J Bacterio189, 706-71 I 18 Miller HI, Friedman DI (1980) An E coli gene product required for site specific recombination. Cell 20, 71 !-719 19 Kitakawa M, lsono K (1982) An amber mutation in the gene rpsA for ribosomal protein S! in Escherichia coli. Mol Gen Genet 185,445--447 20 Van Haute E Joos H, Maes M, Warren G, Van Montagu M, Schell J {1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of AgnJbacterium nemefaciens. EivJBO J 2, 41 I-417 21 Simon R, Priefer U, Ptihler A c 1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative I~acteria. Biotechnology I, 784-79 I 22 Reverchon S, Nasser W. Robert-Baudouy J (1991) Characterization of kdgR, a gene of Erw#da chrysanthemi that regulates pectin degrauation. Mol Micmbiol 5, 2203-2216 23 Mead DA, Szczesna-Skorupa E, Kemper B (1986) Single-stranded DNA 'blue" T7 promoter plasmids: a versatile tandem pJ'omuter system for cloning and protein engineering. Proteb~ Eng I. 67-74 24 Jones JDG, Gutterson N (1987) An efficient mobilizable cosmid vector pRK7813, and its use in a rapid method for marker exchange in Pseudomonasfluorescens strain HV37a. Gene 61,299-306 25 Lemer CG, Inouye M (1990) Low copy number plasmids for regulated lowlevel expression of cloned genes in Escherichia coli with blue/white insert screening capability. Nucleic Acids Res ! 8, 463 I 26 Mise K (1971) Isolation and characterization of a new generalized transducing bacteriophage different from PI in Escherichia coli. J t'~rol 7. 168175 27 R~sibois A, Colet M, Faelen M, Schoonejans E, Toussaint A (1984) @EC2. a new generalized transducing phage of Erwinia chrysanthemi. Virohpgy 137, 102-112

1062 28 Miller JH (1972) Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Hadx)r, New York 29 Glansdorff N (1965) Topography of cotransductible arginine mutation in E coil KI2. Genetics 51,167-17.0 30 Arber W, Enquist L, Holm B, Murray N, Murray K (1983) Experimental methods for use with lambda. In: Lambda Ii (Hendrix RW, Roberts JW. Stahi FW, Weigher8 RA, eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 433 p 31 Bukhari AI, Ljuagquist E (1977) Bacteriophage Mu: methods for cultivation and use. In: DNA Insertion Elements. Plasmids and Episomes (Bukhari AI. Shapiro JA, Adhya SL, eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 749 p 32 Toussaint A, Desmet L, Faelen M, Alazaxd R, Chandler M, Pato M (1987) In vivo mutagenesis of bacteriopbage Mu transposase. J Bacwriol 169, 570057O7 33 Franza 1". Enard C, van Gijscgem F, Expert D (1991) Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron-transport system: characterization of a gen¢ cluster involved in uptake and biosynthetic pathways. Mol Microbiol 5, 1319-1329 34 Ish-Horowicz D, Burke J (1981) Rapid and efficient cosmid vector cloning. Nucleic AcMs Rt's o+ 2q89-2999 35 Maniatis T, Frilsch EF, Sambmok J (1982) Molecular Cloning: A Laboratory. Manual. Cold Spring Harbor Laboratory Pre~. Cold Sprinp. Harbor. New York 36 Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395 37 Fayat G, Mayaux JF, Sacerdot M, From.ant M, Springer M, GranbergManago M, Blanquet S (1983) Excherichia co~~ pbenylalanyl-tRNA synthetase opemn r~.~:ion. Evidence for an attenuation mechanism. Identification of the gene for the ribosomal protein L20. J Mol Biol 171,239-261 38 Friedrich MJ, Deveaux LC, Kedner RJ (1986) Nncleotide sequence of the bIuCED genes involved in vitamins Bi2 transport in Escherichia coil and homology with components of periplasmic-binding-pmtein.dependent transport systems. J Bacteriol 167, 928-934 39 Mechulam Y, Fayat G, Blanquet S (1985) Sequence of the Escherichia ,off pheST operon and identification of the hinut gene. J Bacteriol 163, 787-791 40 Plumbridge JA, Springer M (1980) Genes for the two subonits of phenylalanyl-tRNA synthetase of Escherichia coil are transcribed from the same promoter. J Mol Biol 144, 595-596

41 Sanger F. Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Nail Acad Sci USA 74. 5463-5467 42 Miller H! (1984) Primary structure of the himA gene of Escherichia to//: homology with DNA-bmding protein HU and association with the phenylalanyl-tRNA synthelase opemn. Cold Spring Harbor Syrup Quant Biol 49. 691-698 43 Mechulam Y, Blanquel S, Fayat G (1987) Dual level control of the Escherichia coil pheST-himA operon expression, tRNAt~-dependent attenuation and tmP.scriptional operator-repressor control by himA and the SOS network. J Mol Blot 197. 453-470 44 Ramm EL. Weisbetg RA (1985) Primary structure of the hip gene of Excherichia co~~ and of its product, the sulmmit of integration host factor. J Mol Bio1183. !17-128 45 Gamas P. Burger AC, Churchward G. "Cam L. Galas UP,Chandler M (1986) Replication of pSCIOi: effects of mutations in the E coli DNA binding protein IHE Mol Gen Genet 204, 85-89 46 Prentki P, Krisch H (1984) In t.itro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303-313 47 El Hassouni M, Hemissat B. Chippaux M. Barras F (1992) Nucleotide sequence of the arb genes, which control [$.81ucoside utilization in Erwinia chrysanthemi: comparison with the Escherichia coli bgl operon and evidence for a new 13-glycohydrolase family including enzymes from Eubucteria+ Archeabac!eria, and humans. J Bacteriol 174, 765-777 48 Schnetz K, Toloczyki C. Rak B (1987) l~431ucoside (bgl) operon of Escherichia co~~K- 12: nucleotide sequence, genetic organization, and possible evolutionary relationship to regulatory components of two Bacillus suhti//s genes, J Bacteriol 169, 2579-2590 49 Danchin A. Lenzen G (1988) Structure and evolution of bacterial adenylate cyclase: comparison between Excherichia coil and Erwinia chrysanthemL Se Mess Phosphoprot 12, 7-28 50 DeFeyter RC, David~n BE, Pittard J (1986) Nncleotide .~-quence of the transcription unit containing the aroL and atom genes from Escherichia c~di K-12. J Bm'teriol 165, 233-239 i 51 Minton NP, Whitehead PJ, Atkinson 1", Gilbert HJ (1989) Nncleotide sequence of an Erwhda chrysanthemi gene encoding shikimate kinase. Nucleic Acids Res 17, 1769-1769 52 Drlica K, Rouvi~re-Yaniv J (1987) Histonelike protein of bacteria. Microbiol Rer 51, 301-319