To facilitate efficient allelic exchange of genetic information into a wild-type strain background, we improved upon and merged approaches using a temperature-sensitive plasmid and a counter-selectable marker in the chromosome. We first constructed intermediate strains of Escherichia coli K12 in which we replaced wild-type chromosomal sequences, at either the fimB-A or lacZ-A loci, with a newly constituted DNA cassette. The cassette consists of the sacB gene from Bacillus subtilis and the neomycin (kanamycin) resistance gene of Tn5, but, unlike another similar cassette, it lacks IS1 sequences. We found that sucrose sensitivity was highly dependent on incubation temperature and sodium chloride concentration. The DNA to be exchanged into the chromosome was first cloned into derivatives of plasmid pMAK705, a temperature-sensitive pSC101 replicon. The exchanges were carried out in two steps, first selecting for plasmid integration by standard techniques. In the second step, we grew the plasmid integrates under non-selective conditions at 42 degrees C, and then in the presence of sucrose at 30 degrees C, allowing positive selection for both plasmid excision and curing. Despite marked locus-specific strain differences in sucrose sensitivity and in the growth retardation due to the integrated plasmids, the protocol permitted highly efficient exchange of cloned DNA into either the fim or lac chromosomal loci. This procedure should allow the exchange of any DNA segment, in addition to the original or mutant allelic DNA, into any non-essential parts of the E. coli chromosome.

the pr0duct 0f the rp0H (htpR) 9ene, 0 ~2, d1rect5 RNA p01ymera5e t0 1n1t1ate tran5cr1pt10n fr0m heat 5h0ck pr0m0ter5 at a11 temperature5. 7ran5cr1pt10n 0f the heat 5h0ck 9ene5 15 1ncrea5ed when ce115 are exp05ed t0 h19h temperature5 6ecau5e 0f 1ncrea5ed tran5cr1pt10n 1n1t1at10n 6y cr32-RNA p01ymera5e. A5 a 5tep t0ward under5tand1n9 the re9u1at10n 0f the heat 5h0ck re5p0n5e we have exam1ned the tran5cr1pt10n 0f the rp0H 9ene. U51n9 51 mapP1n9, pr0m0ter c10n1n9, and 1n v1tr0 tran5cr1pt10n, we have 1dent1f1ed the pr0m0ter5 and the term1nat0r f0r the rp0H tran5cr1pt10n un1t. 7he rp0H tran5cr1pt5 are m0n0c15tr0n1c and 0r191nate fr0m at 1ea5t three pr0m0ter5. N0ne 0f the pr0m0ter5 15 rec09n12ed 6y cr32-RNA p01ymera5e. 7w0 are rec09n12ed 6y (r7°-RNA p01ymera5e and are act1ve at 60th 10w and h19h 9r0wth temperature5. We d0 n0t kn0w what f0rm 0f RNA p01ymera5e rec09n12e5 the th1rd pr0m0ter. 7ran5cr1pt5 fr0m th15 pr0m0ter are a6undant 0n1y at h19h temperature and are pre5ent after 5h1ft t0 the 1etha1 temperature 0f 50°C, even at t1me5 when there are n0 detecta61e tran5cr1pt5 fr0m the 0ther rp0H pr0m0ter5. 7he am0unt 0f rp0H mRNA 1ncrea5e5 f1vef01d 6y 8 m1n after 5h1ft fr0m 30 t0 43.5°C 6ut rp0H mRNA 5ynthe515 1ncrea5e5 6y 1e55 than tw0f01d, 1nd1cat1n9 that there 15 p05t-tran5cr1pt10na1 c0ntr01 0f the 1eve1 0f rp0H mRNA and pre5uma61y 0f cr 32.[Key W0rd5:P05t-tran5cr1pt10na1 re9u1at10n; heat 5h0ck; 519ma-32; rp0H; htpR] When ce115, t155ue5, 0r even wh01e 0r9an15m5 are exp05ed t0 e1evated temperature5 they re5p0nd 6y 1n-crea51n9 the 5ynthe515 0f the heat 5h0ck pr0te1n5. 7h15 re5p0n5e t0 h19h temperature, termed the heat 5h0ck re5p0n5e, 15 51m11ar 1n 0r9an15m5 fr0m every 6101091ca1 k1n9d0m (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 1f ce115 are 5h1fted fr0m a 10w temperature t0 a h19her 0ne w1th1n the1r 9r0wth ran9e, the 1nduct10n 0f the heat 5h0ck pr0te1n5 15 tran51ent. 1f the ce115 are tran5ferred t0 temperature5 a60ve the1r 9r0wth ran9e, the heat 5h0ck pr0te1n5 are expre55ed at h19h 1eve15 f0r a5 10n9 a5 the ce115 5ynthe512e pr0te1n (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 7he funct10n5 0f the heat 5h0ck pr0te1n5 are n0t we11 under5t00d. 1t 15 c1ear that 50me 0f the heat 5h0ck pr0-te1n5 are re4u1red f0r 5urv1va1 at h19h temperature and that expre5510n 0f the heat 5h0ck pr0te1n5 1ncrea5e5 the 1en9th 0f t1me ce115 5urv1ve at temperature5 t00 h19h f0r 9r0wth (Ne1dhardt et a1. 1984; L1nd4u15t 1986). 5t1mu11 0ther than heat can 1nduce the 5ynthe515 0f var10u5 heat 5h0ck pr0te1n5 (Ne1dhardt et a1. 1984; L1nd4u15t 19861. 7he5e 5t1mu11 1nc1ude 5tarvat10n f0r nutr1ent5, v1ra1 1nfect10n, and exp05ure t0 ethan01, 0x1dant5, UV rad1a-t10n, am1n0 ac1d ana1095, 0r DNA-dama91n9 a9ent5. 1t 5eem5 that the heat 5h0ck pr0te1n5 he1p pr0tect ce115 fr0m a var1ety 0f adver5e c0nd1t10n5. 7he heat 5h0ck re5p0n5e 1n E5cher1ch1a c011151nduced rap1d1y. 7he 5ynthe515 0f the 20 0r 50 heat 5h0ck pr0te1n5 15 1ncrea5ed w1th1n 1 m1n after 5h1ft fr0m 30°C t0 a h19her 9r0wth temperature, 5uch a5 43°C (Ne1dhardt et a1. 1984). Max1mum rate5 0f heat 5h0ck pr0te...

No abstract

We have sequenced a cloned segment of E. coli chromosomal DNA that includes the heat shock regulatory gene htpR. This segment contains an 852 nucleotide open reading frame bounded by transcriptional and translational signals. Both in vivo and in vitro the cloned segment produces a single protein that migrates in gels with the cellular protein (F33.4) implicated as the htpR product. Properties of a cloned fragment of the coding sequence truncated at the promoter-distal end are consistent with this assignment. The htpR gene product appears homologous to the sigma factor of RNA polymerase, and the two proteins are predicted to have similar secondary structure. In addition, two regions of the predicted htpR product resemble protein-DNA contact points conserved in known DNA-binding proteins.

The on-and-off expression (phase variation)of type 1 fimbriae, encoded by fimA, in Escherichia coli is controlled by the inversion of a promoter-containing 314-basepair DNA element. This element is flanked on each side by a 9-base-pair inverted, repeat sequence and requires closely linked genes for inversion. Homology analysis of the products of these genes, fimB andfimE, reveals a strong similarity with the proposed DNA binding domain of X integrase, which mediates site-specific recombination in the presence of integration host factor. Integration host factor, encoded by himA and hip/himD, binds to the sequence 5' TNYAANNNRTTGAT 3', where Y = pyrimidine and R = purine, in mediating integration-excision. In analyzing the DNA flanking the fim 314-base-pair inversion sequence, we found the adjacent sequence 5' TTTAACTTATTGAT 3', which corresponds perfectly with the consensus integration host factor binding site. To characterize the role of himA in phase variation, we transduced either a deletion of himA or an insertionally inactivated hip/himD gene into an E. coli strain with a fimA-acZ operon fusion. We found the rate of phase variation decreases sharply from 10-3 to <10-5 per cell per generation. Southern hybridization analysis demonstrates that the himA mutation results in a failure of the switch-generated genetic rearrangement. When the transductant was transformed with a himA+ plasmid, normal switching returned. Thus integration host factor is required for normal type 1 runbriae phase variation in E. coli.Genome rearrangement mediated by site-specific recombination is of widespread importance in the control of gene expression in prokaryotes and eukaryotes (1). The molecular basis for the mechanisms has been determined in several systems, including bacteriophage integration-excision (2, 3) and the interrelated invertible DNA elements best exemplified by the flagellar switch in Salmonella (4-6). In both systems the following three sets of factors are required. (i)The cis-specific DNA that acts as the crossing point for the rearrangement.(ii) Site-specific trans-active factors whose genes map near the cis-specific DNA. For example, in X integration the int gene is carried by the bacteriophage (7-9), and in the Salmonella switch the hin gene is located within the invertible DNA (5). (iii) "Host factors" whose genes reside on the bacterial chromosome distant from the site of rearrangement. The best studied host factor is the integration host factor (IHF) of Escherichia coli required for X phage integration (10). IHF consists of two subunits: IHFa, a Mr 10,500 polypeptide encoded by himA that maps at 38 min, and IHF,B, a Mr 9500 polypeptide encoded by hip/himD that maps at 20 min (11)(12)(13)(14). Like Salmonella flagellar expression, type 1 fimbriae of E. coli exhibit phase-variation control. We have shown that E. coli phase variation is transcriptionally regulated (15) and that the oscillating expression is due to the specific inversion of a 314-base-pair (bp) invertible element of DNA that directs transc...