Mutation Research, 264 (1991) 93-96

93

© 1991 Elsevier Science Publishers B.V. All rights reserved. 0165-7992/91/$03.50 ADONIS 0165799291000905 MUTLET 0535

Location of a mutation in the aspartyl-tRNA synthetase gene of Escherichia coli K 12 Gary J. Sharpies and Robert G. Lloyd Department of Genetics, University of Nottingham, Medical School, Queens Medical Centre, Nottingham, NG7 2UH (Great Britain) (Received 23 May 1991) (Revision received 24 June 1991) (Accepted 24 June 1991)

Keyworda: Aspartyl-tRNA synthetase gene; Tls-1 mutation; (E. coli K12)

Summary A mutation (tls-1) that confers a temperature-sensitive growth phenotype in Escherichia coli was shown by DNA cloning and sequencing to be an allele of aspS, the gene for aspartyl-tRNA synthetase. The mutation, which lies near minute 41 on the genetic map, was located some 2.3 kb from the 5' end of the ruvAB operon. A DNA fragment encoding the carboxy-terminus of AspRS was found to be sufficient to allow growth of a tls-1 strain at the non-permissive temperature.

The tls-1 mutation of Escherichia coli Kl2 causes a temperature-sensitive growth phenotype in low salt media (Attfield et al., 1985). It was identified during a screening for ruv mutants deficient in DNA repair after hydroxylamine mutagenesis of the eda region (Shurvinton et al., 1984; C.E. Shurvinton, Ph.D. Thesis, University o f Nottingham, 1983). Genetic crosses revealed that the mutation is 40-50°/0 cotransducible with eda and lies very close to ruv in the order tls-ruv-eda (C.E. Shurvinton, L.R. Overton and R.G. Lloyd, unpublished results). We have cloned a DNA fragment from the Correspondence: Professor Robert G. Lloyd, Department of Genetics, University of Nottingham, Medical School, Queens Medical Centre, Nottingham, NG7 2UH (Great Britain), Tel. 0602 709406; Fax 0602 422225.

vicinity of the ruv region that alleviates the temperature-sensitivity of tls-I strains and determined its nucleotide sequence. The results are presented here since they show that tls-1 is an allele of aspS, the previously unlocated gene for aspartyl-tRNA synthetase. In our studies, we made use of strain CS89, a tls-1 eda-51::TnlO derivative of AB1157 [F- thi-1 hisG4 A(gpt-proA)62 argE3 thr-1 leuB6 kdgK51 rfbD1 ara-14 lacY1 galK2 xyl-5 mtl-1 supE44 tsx-33 rpsL31 (Bachmann, 1987)]. CS89 (tls-1 eda-51::TnlO) was made by transducing AB 1157 to tetracycline resistance with a phage Plvir stock grown on a strain carrying tls-1 linked to eda-51::TnlO (C.E. Shurvinton, Ph.D. Thesis, University of Nottingham, 1983). The presence of tls-I prevents cells from forming colonies after in-

94

cubation overnight at 42°C on LB agar containing sodium chloride at a concentration of 0.5 g/1 (Lloyd et al., 1974). However, growth is not fully inhibited at 42°C. The data shown in Fig. 1 demonstrate that strain CS89 continues to grow after a shift from 30°C to 42°C, though at a much reduced rate. Division is not inhibited, with the result that the cells become shorter as incubation proceeds at the non-permissive temperature. The ceils remain viable and on LB agar form visible colonies within 4-5 days. When the salt concentration in the LB media was increased to 10 g/l, CS89 was able to grow almost as well as the tls ÷ control strain AB1157 (data not shown). Three-factor, P1 transductional crosses located tls very close to the ruv region of the chromosome, which has been cloned and dissected in some detail (Attfield et al., 1985; Sharpies et al., 1990). From the linkage data (not shown), tls was expected to lie 5' of the r u v A B operon (see Fig. 2). Overlapping restriction fragments covering this region were subcloned into pBR322 (Bolivar et al., 1977) and pUC18 (Yanisch-Perron et al., 1985), using pPVA101 (Attfield et al., 1985) as the primary source of DNA. The constructs made were transformed into strain CS143 (tls-1), a tetracycline-sensitive derivative of CS89, to see if they were able to restore normal growth at 42°C. The results obtained (Fig. 2) demonstrated that plas-

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Incubation time (h) Fig. 1. Effect of temperature on the growth of tls-1 and tls + strains. At time zero, samples of overnight cultures of CS89 and AB1157 were inoculated into low salt LB broth and incubated at 30°C until the A65o reached about 0.1 after which half the culture was transferred to 42°C. Growth was then monitored at both temperatures over the period indicated.

mids carrying the 0.5 kb PstI(1)-HinclI fragment allow CS143 to grow well at 42°C. We conclude that tls-1 must be located within the corresponding P s t I ( l ) - H i n c l I interval of the chromosome. The nucleotide sequence of the 1084 base pair B a m H I - H i n c l I region of pPVA101 was determined and is shown in Fig. 3. Database searches revealed that the sequence from the P v u l I site at bp 146 to the S a u 3 A I site at bp 297 is identical to a se-

11 pHSG415

orf-33 ruvC

ruvA r u v B

Ability to restore normal growth at 42°C to CS143 pFB512

+

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Fig. 2. Structure of recombinant plasmids carrying the t/s and ruv regions of the E. coli chromosome and complementation analysis of tls-1. The organisation of the DNA insert in pPVA101 and pFB512 is taken from Attfield et al. (1985), Benson et al. (1988) and Sharpies et al. (1990). The regions in pPVA101 between H i n d l l l ( 1 ) and B a m H l and between r u v B and Pstl(2) are abbreviated for economy of presentation. The heavy dashed line denotes a sequence that is not contiguous with the ruv region in the chromosome. Restriction sites are labelled with vertical arrows and only those used for insertion of the cloned DNA into the vectors are shown. Sites labelled with an asterisk are not necessarily unique. The dashed vertical lines are used to indicate the alignment of the cloned sequences. Vector sequences are identified below each construct and are not to scale. The ability to complement tls-I was determined by transforming the test plasmid into strain CS143 at 30°C and then examining the ampicillin-resistant transformants for their ability to grow at 42°C ( + , growth; - , no growth).

95

quence from the prs region located at minute 21 on the chromosomal map (Hove-Jensen et al., 1986). This observation confirms previous indications from Southern analyses that the DNA insert in pPVA101 (Fig. 2) from the HindlII(1) site through to a point between the B a m H I and PstI(1) sites is not contiguous in the chromosome with the PstI(1)-HindlII(2) region containing the ruv genes (F.E. Benson, Ph.D. Thesis, University of Nottingham, 1988). The 10.5 kb HindlII insert in pPVA101 was subcloned from the kruv + transducing phage, RLI01 (Shurvinton et al., 1984), which came from a library generated using a partial Sau3AI digest of chromosomal DNA (Arthur et al., 1982). The presence of non-contiguous sequences in pPVA101 is therefore not too surprising. The 144-bp sequence from the Sau3AI site starting at bp 2 to the PvuII site at bp 146 contains BamHI

GGATCCCCGACAGC~GGGTCGC~CCAGCAGTCCTGCCAGTCCAAACTG TTGCGACACGGGATAGAG~TTACCACAAATGCCGCAGTCGGACCGGAAA CGCTA~GCGTGACCCACCCGTCAGAGC~TGAC~TCCCCGC~CAGCT GCGGTATATA~CCGTACTGGGGTGCCACACCACTACC~TAGCC~CGC CATCGCCAGCGGGATAGC~T~TCCCGACGGTTATCCCG~TCAGGT CACGGGTAAACCGTGCGGCAGTATATTTTTCTTTC~GC~GCGTCGATC AAAG~GAG~TATCCAG~GCTCACTATTGAT~CTACCGCGAGCATGA D TCGTACTGCCGCGC~GATGGCGATATGATTTTCTTCGGTGCCGAC~CA R T A A Q D G D M I F F G A D N K AGAAAATTGTTGCCGACGCGATGGGTGCACTGCGCCTGAAAGTGGGTAAA K I V A D A M G A L R L K V G K GACCTTGGTCTGACCGACGAAAGCAAATGGGCACCGCTGTGGGTTATCGA D L G L T D E S K W A P L W V I D CTTCCCGATGTTTG~GACGACGGTG~GGCGGCCTGACGGC~TGCACC F P M F E D D G E G G L T A M H H

50 100 150 200 250 300 350 400 450 500 550

PstI

ATCCGTTCACCTCACCGAAAGACATGACGGCTGCAG~CTGAAAGCTGCA P F T S P K D M T A A E L K A A CCGGAA.%ATGCGGTGGCG~CGCTTACGATATGGTCATC~TGGTTACGA P E N A V A N A Y D M V I N G Y E AGTGGGCGGTGGTTCAGTACGTATCCAT~TGGTGATATGCAGCAGACGG V G G G S V R I H N G D M Q Q T V TGTTTGGTATTCTGGGTATC~CG~GAGG~CAGCGCGAGAAATTCGGC F G I L G I N E E E Q R E K F G TTCCTGCTCGACGCTCTGA~TACGGTACTCCGCCGCACGCAGGTCTGGC F L L D A L K Y G T P P H A G L A ATTCGGTCTTGACCGTCTGACCATGCTGCTGACCGGCACCGAC~TATCC F G L D R L T M L L T G T D N I R GTGACGTTATCGCCTTCCCGA~CCACGGCGGCAGCGTGTCTGATGACT D V I A F P K T T A A A C L M T G~GCACCGAGCTTTGCT~CCCGACTGCACTGGCTGAGCTGAGCATTCA E A P S F A N P T A L A E L S I Q GGTTGTG~G~GGCTGAG~T~CTGATATGACTCAAATACACGAAATC V V K K A E N N

600 650 700 750 800 850

Acknowledgements

900 950 1000

ATTCGCGTTGCATCGAGGCGGC~CTGAGTG~CTCCCATGAGCATAGAT 1050 HincII

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no internal Sau3AI site and therefore is presumably contiguous with the prs sequence in the chromosome. The small Sau3AI fragment from bp 297-349 is of unknown origin. The sequence from bp 349-1026 was found to match perfectly the 3' end of the structural gene for aspartyl-tRNA synthetase, the complete sequence of which has been published recently (Eriani et al., 1990). The region downstream of this sequence contains part of a 126-bp inverted repeat unit (IRU) found in the genomes of several bacterial species (Sharpies and Lloyd, 1990). Since multicopy plasmids carrying the 500 bp PstI(1)HinclI fragment allow a tls-1 strain to grow well at 42°C, and aminoacyl-tRNA synthetase are essential for growth, we conclude that tls-1 is an allele of aspS. We assume that the carboxyterminal fragment of the AspRS protein is sufficient to restore tRNA synthetase activity. The ability of portions of aminoacyl-tRNA synthetases to complement mutant phenotypes has been observed previously (Jasin et al., 1984, 1985). The carboxyterminal fragment of AspRS identified here lacks some of the sequence motifs thought to be needed for activity (Eriani et al., 1990). However, it is possible that a heterodimer containing the carboxyterminal fragment and the mutant tls-1 product has some activity. In conclusion, the tls-1 mutation locates aspS to minute 41 on the genetic map. No other mutation in aspS has been reported (Eriani et ah, 1990). The availability of tls-1 will facilitate the functional analysis of AspRS.

1084

Fig. 3. Nucleotide sequence of the 1084-bp B a m H I - H i n c l l region of the D N A insert cloned in pPVA101. Relevant restriction enzyme sites used are marked above the D N A sequence. The translation of the 3' end of the a ~ S reading frame is shown below the D N A sequence. The accession number for the t& sequence is X53984. The accession number for the sequence adjoining prs is X53983.

We thank L.R. Overton for her help with some of the experiments and Carol Buckman for technical support. This work was supported by grants to R.G.L. from the Science and Engineering Research Council, The Medical Research Council, and The Wellcome Trust.

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96 Attfield, P.V., F.E. Benson and R.G. Lloyd (1985) Analysis of the ruv locus ofEscherichia coli K-12 and identification of the gene product, J. Bacteriol., 164, 276-281. Bachmann, B.J. (1987) Derivations and genotypes of some mutant derivatives of Escherichia coil K-12, in: F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter and H.E. Umbarger (Eds.), Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, American Society for Microbiology, Washington, DC, pp. 1190-1219. Benson, F.E., G.T. llling, G.J. Sharpies and R.G. Lloyd (1988) Nucleotide sequencing of the ruv region of Escherichia coli K-12 reveals a LexA regulated operon encoding two genes, Nucl. Acids Res., 16, 1541-1549. Bolivar, F., R.L. Rodriguez, P.J. Greene, M.C. Betlach, H.L. Heynecker, H.W. Boyer, J.H. Crosa and S. Falkow (1977) Construction and characterization of new cloning vehicles, 11. A multipurpose cloning system, Gene, 2, 95-113. Eriani, G., M. Delarue, O. Poch, J. Gangloff and D. Moras (1990a) Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs, Nature (London), 347, 203-206. Eriani, G., G. Dirheimer and J. Gangloff (1990b) Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases, Nucl. Acids Res., 18, 7109-7118. Hove-Jensen, B., K.W. Harlow, C.J. King and R.L. Switzer (1986) Phosphoribosyl pyrophosphate synthetase of Escherichia coli, J. Biol. Chem., 261, 6765-677L

Jasin, M., L. Regan and P. Schimmel (1984) Dispensable pieces of an aminoacyl-tRNA synthetase which activate the catalytic site, Cell, 36, 1089-1095. Jasin, M., L. Regan and P. Schimmel (1985) Two mutations in the dispensable part of alanine-tRNA synthetase which affect the catalytic activity, J. Biol. Chem., 260, 2226-2230. Lloyd, R.G., K.B. Low, N.G. Godson and E.A. Birge (1974) Isolation and characterization of an Escherichia coli K-12 mutant with a temperature-sensitive r e c A - phenotype, J. Bacteriol., 120, 407-415. Sharpies, G.J., and R.G. Lloyd (1990) A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes, Nucleic Acids Res., 18, 6503-6508. Sharpies, G.J., F.E. Benson, G.T. llling and R.G. Lloyd (1990) Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination, Mol. Gen. Genet., 221,219-226. Shurvinton, C.E., R.G. Lloyd, F.E. Benson and P.V. Attfield (1984) Genetic analysis and molecular cloning of the Escherichia coli ruv gene, Mol. Gen. Genet., 194, 322-329. Yanisch-Perron, C., J. Vieira and J. Messing (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors, Gene, 33, 103-119. Communicated by R.P.P. Fuchs