Complementation of a trpE deletion in Escherichia coli by Spirochaeta aurantia DNA encoding anthranilate synthetase component I activity.

A 2.7-kilobase Sau3A fragment of Spirochaeta aurantia DNA cloned in pBR322 complemented a trpE deletion in Escherichia coli. Deletion analysis and Tn5 mutagenesis of the resulting plasmid pBG100 defined a 2-kilobase-pair region that was required for both the complementation and the synthesis of 59,000- and 47,000-molecular-weight polypeptides (59K and 47K polypeptides) in maxicells. Both the 59K and the 47K polypeptides appear to be encoded by a single gene. A maxicell analysis of pBG100::Tn5 mutants suggests that the 47K polypeptide is not sufficient for the trpE complementation. In vitro and in vivo anthranilate synthetase (AS) assays indicate that the complementing activity encoded by pBG100 was functionally analogous to the AS component I of E. coli in that it utilized NH3 but not glutamine as the amino donor. pBG100 did not encode a glutamine amidotransferase activity, although the AS component I it encoded was capable of interacting with E. coli AS component II to catalyze the glutamine-requiring reaction. Expression appeared to depend on a promoter in the cloned S. aurantia DNA.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: II. Cloning and expression in Escherichia coli

The genes for the large and small subunits of anthranilate synthase (trpE and trpG, respectively) have been cloned from Pseudomonas aeruginosa PAC174 into E. coli by R-prime formation with the broad-host- range plasmid R68.44. Sequential subcloning into plasmid vectors reduced the active Pseudomonas DNA fragment to a length of 3.1 kb. We obtained evidence that this region contains the promoter for its own expression and retains a vestigial regulatory response to tryptophan scarcity or excess.      

Complementation of mutations in Escherichia coli and Bordetella pertussis by B. pertussis DNA cloned in a broad-host-range cosmid vector

A gene library of Bordetella pertussis DNA was constructed in Escherichia coli using the broad-host-range cosmid vector pLAFR1. The average insert size was 24.9 kb. From 500 members of the gene library, clones were identified which complemented trpE, glnA and Thr- mutations in E. coli but none which complemented trpD, trpC, trpB, trpA, proA or Leu- mutations. Four clones were identified which complemented trpE in E. coli. Anthranilate synthase activity was detected in a trpE strain only when it harboured a plasmid from one of these clones; activity was repressed when tryptophan was included in the growth medium. Two clones were identified which complemented glnA of E. coli. A recombinant plasmid from one of these clones also restored some of the nitrogen acquisition functions of glnG and glnL in E. coli. Expression of several B. pertussis virulence-associated products (haemolysin, heat-labile toxin, adenylate cyclase, filamentous haemagglutinin, and the cell-envelope polypeptide of Mr 30,000) was not detected in 500 independent clones. However, by transferring the recombinant plasmids to a mutant of B. pertussis deficient in haemolysin and adenylate cyclase, a plasmid was identified which restored both these activities.     

Cloning and characterization of Escherichia coli K-12 regulator gene tyrR.

The Escherichia coli K-12 regulator gene tyrR was cloned into the multicopy plasmid pBR322 from a lambda(Tn10)tyrR+ specialized transducing phage. Further subcloning localized the gene within a 2.1-kilobase region. Analysis of plasmid-coded proteins showed that the tyrR gene codes for a 63,000-dalton polypeptide.     

Identification of OmpR: a positive regulatory protein controlling expression of the major outer membrane matrix porin proteins of Escherichia coli K-12.

We report here on the cloning of a gene located within the ompB locus of E. coli K-12. This gene, designated ompR, resides on a 10.9-kilobase EcoRI fragment that we cloned, using a lambda vector and in vitro packaging techniques. By subcloning portions of this fragment into the high-copy-number plasmid pBR322, we have isolated the ompR gene on a 1.4-kilobase EcoRI-AvaI fragment. This fragment has been characterized physically and will facilitate a more detailed study of the role and mechanism of porin regulation by the ompB locus.     

Dihydroorotase from Escherichia coli. Cloning the pyrC gene and production of tryptic peptide maps

We have inserted a 1.7-kilobase pair Escherichia coli DNA fragment containing the 1-kilobase pair pyrC gene into the high copy number plasmid pKC16. Dihydroorotase expressed by the pyrC plasmid in E. coli constituted 6.3% of the soluble protein in frozen cell paste. Pure dihydroorotase derived from this frozen cell paste was compared with pure enzyme derived from an E. coli strain lacking the pyrC plasmid: tryptic peptide maps from the two dihydroorotase preparations, produced using reverse-phase high performance liquid chromatography, were indistinguishable. We conclude that the entire pyrC gene is present on the hybrid plasmid and that the dihydroorotase produced from this plasmid is identical to the wild type.     

Genetic analysis of the gentamicin resistance region of pPH1JI and incorporation into a wide host range cloning vehicle

A region of the IncP plasmid pPH1JI encoding resistance to gentamicin, spectinomycin, and streptomycin was characterized by subcloning, deletion, and insertion mutagenesis. Approximate locations of these resistance determinants were established. A 1.6-kb HindIII-SphI segment of this region expresses gentamicin resistance (Gmr) in Escherichia coli when inserted into various plasmid vectors; this DNA segment encodes a polypeptide of 17.5 kDa. Incorporation of this fragment into an IncP cloning vehicle produced a Gmr wide host range vector, pRAR209, which confers levels of Gmr comparable to those expressed by pPH1JI in E. coli, Agrobacterium tumefaciens, and Rhizobium meliloti.     

Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli.

The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane. A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids. Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region. Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment. The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane. This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12. Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor. A deletion at the 3' end of btuB resulted in formation of an altered receptor. Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins. Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.

The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication).     

Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli.

The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized. A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe. A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe. The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin. The tetanus DNA was expressed in E. coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene. The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.     

Identification, genetic analysis and DNA sequence of a 7.8-kb virulence region of the Salmonella typhimurium virulence plasmid

The 90-kilobase (kb) virulence plasmid of Salmonella typhimurium is responsible for invasion from the intestines to mesenteric lymph nodes and spleens of orally inoculated mice. We used Tn5 and aminoglycoside phosphotransferase (aph) gene insertion mutagenesis and deletion mutagenesis of a previously identified 14-kb virulence region to reduce this virulence region to 7.8kb. The 7.8-kb virulence region subcloned into a low copy-number vector conferred a wild-type level of splenic infection to virulence plasmid-cured S. typhimurium and conferred essentially a wild-type oral LD50. Insertion mutagenesis identified five loci essential for virulence, and DNA sequence analysis of the virulence region identified six open reading frames. Expected protein products were identified from four of the six genes, with three of the proteins identified as doublet bands in Escherichia coli minicells. Three of the five mutated genes were able to be complemented by clones containing only the corresponding wild-type gene. Only one of the five deduced amino acid sequences, that of the positive regulatory element, SpvR, possessed significant homology to other proteins. The codon usage for the virulence genes showed no codon bias, which is consistent with the low levels of expression observed for the corresponding proteins. Consensus promoters for several different sigma factors were identified upstream of several of the genes, whereas only consensus Rho-dependent termination sequences were observed between certain of the genes. The operon structure of this virulence region therefore appears to be complex. The construction of the cloned 7.8-kb virulence region and the determination of the DNA sequence will aid in the further genetic analysis of the five plasmid-encoded virulence genes of S. typhimurium.     

Molecular analysis of the Escherichia coli recO gene.

The plasmid pLC7-47, which contains lep, rnc, and era, was found to complement the UV-sensitive and recombination-deficient phenotypes caused by the recO1504::Tn5 mutation. Southern blotting analysis demonstrated that pLC7-47 contained a segment of Escherichia coli DNA that covered the region of the E. coli chromosome containing the recO1504::Tn5 mutation. A combination of deletion mapping and insertional mutagenesis localized the recO-complementing region to an approximately 1-kilobase region of a 1.6-kilobase BamHI fragment. The DNA sequence of the 1.6-kilobase BamHI fragment was determined and contained part of era and a 726-base-pair recO open reading frame. The recO open reading frame contained three possible translation start codons and could potentially encode a polypeptide of Mr 26,000. Computer analysis indicated that the putative RecO protein had suboptimal codon usage and did not show significant homology with previously identified proteins whose sequences were present in protein data bases. A combination of primary sequence analysis and secondary structure predictions suggested that recO contains a mononucleotide-binding fold.     

Cloning and expression of the rfe–rff gene cluster of Escherichia coli

We have cloned a 13 kb Escherichia coli DNA fragment which complemented the rfe mutation to recover the biosynthesis of E. coli O9 polysaccharide. Using Tn5 insertion inactivation, the rfe gene was localized at the 1.5 kb HindIII-EcoRI region flanking the rho gene. We constructed an rfe-deficient E. coli K-12 mutant by site-directed inactivation using a DNA fragment of the cloned 1.5 kb rfe gene. This also confirmed the presence of the rfe gene in the 1.5 kb region. By simultaneous introduction of both the rfe plasmid and the plasmid of our previously cloned E. coli O9 rfb into this rfe mutant, we succeeded in achieving in vivo reconstitution of O9 polysaccharide biosynthesis. From sequence analysis of the rfe gene, a putative promoter followed by an open reading frame (ORF) was identified downstream of the rho gene. This ORF coincided with the position of the rfe gene determined by Tn5 analysis and site-directed mutagenesis. Furthermore, we identified the rff genes in the 10.5 kb DNA flanking the rfe gene. We recognized at least two functional domains on this cloned rff region. Region I complemented a newly found K-12 rff mutant, A238, to synthesize the enterobacterial common antigen (ECA). Deletion of region II resulted in the synthesis of ECAs with shorter sugar chains. When the 10.5 kb rff genes of the plasmid were inactivated by either deletion or Tn5 insertion, the plasmid lost its ability to give rise to transformants of the rfe mutants.     

Characterization and mapping of regions encoding clindamycin resistance, tetracycline resistance, and a replication function on the Bacteroides R plasmid pCP1.

The Bacteroides drug resistance plasmid pCP1 encodes clindamycin resistance (Clr) and a cryptic tetracycline resistance (Tcr) determinant that is expressed in Escherichia coli cells grown aerobically, but not anaerobically, and is not expressed phenotypically in Bacteroides spp. Localization of genetic functions on pCP1 was facilitated by the construction of hybrid shuttle plasmids containing portions of pCP1 ligated to pDG5, a pBR322 derivative carrying the RK2 transfer origin. pDP1 delta 4 is a BglII deletion derivative of pCP1 linked to pDG5 and can be maintained in both E. coli and Bacteroides fragilis. By using Tn5 mutagenesis and subcloning, we localized the Clr and Tcr regions on the EcoRI B fragment between the 1.2-kilobase direct repeats of pCP1. The Clr and Tcr determinants are distinct and appear to be transcribed separately. Control of the Tcr phenotype is unusual in that expression is constitutive and is enhanced by a region encompassing the adjacent direct repeat. In addition, a region of pCP1 required for replication in Bacteroides spp. has been identified in the neighboring EcoRI A fragment.     

Isolation and complementation of mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen.

Mutants of Anabaena sp. strain PCC 7120 unable to grow aerobically on dinitrogen were isolated by mutagenesis with UV irradiation, followed by a period of incubation in yellow light and then by penicillin enrichment. A cosmid vector, pRL25C, containing replicons functional in Escherichia coli and in Anabaena species was constructed. DNA from wild-type Anabaena sp. strain PCC 7120 was partially digested with Sau3AI, and size-fractionated fragments about 40 kilobases (kb) in length were ligated into the phosphatase-treated unique BamHI site of pRL25C. A library of 1,054 cosmid clones was generated in E. coli DH1 bearing helper plasmid pDS4101. A derivative of conjugative plasmid RP-4 was transferred to this library by conjugation, and the library was replicated to lawns of mutant Anabaena strains with defects in the polysaccharide layer of the envelopes of the heterocysts. Mutant EF116 was complemented by five cosmids, three of which were subjected to detailed restriction mapping; a 2.8-kb fragment of DNA derived from one of the cosmids was found to complement EF116. Mutant EF113 was complemented by a single cosmid, which was also restriction mapped, and was shown to be complemented by a 4.8-kb fragment of DNA derived from this cosmid.