Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

The serC-aro A operon of Escherichia coli. A mixed function operon encoding enzymes from two different amino acid biosynthetic pathways.

Sub-cloning experiments aimed at precisely locating the E. coli aroA gene, which encodes the shikimate pathway enzyme 5-enolpyruvylshikimate 3-phosphate synthase, showed that in certain constructions, which remain capable of complementing an auxotrophic aroA mutation, expression of aroA is reduced. DNA sequence analysis revealed that a sequence approx. 1200 base pairs (bp) upstream of aroA is necessary for its expression. An open reading frame was identified in this region which encodes a protein of 362 amino acids with a calculated Mr of 39,834 and which ends 70 bp before the start of the aroA coding sequence. This gene has been identified as serC, the structural gene for 3-phosphoserine aminotransferase, an enzyme of the serine biosynthetic pathway. Both genes are expressed as a polycistronic message which is transcribed from a promotor located 58 bp upstream of serC. Evidence is presented which confirms that the aroA and serC genes constitute an operon which has the novel feature of encoding enzymes from two different amino acid biosynthetic pathways.     

Genes aroA and serC of Salmonella typhimurium constitute an operon.

Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon.     

Molecular gene cloning and nucleotide sequencing and construction of an aroA mutant of Pasteurella haemolytica serotype A1.

The aroA gene of Pasteurella haemolytica serotype A1 was cloned by complementation of the aroA mutation in Escherichia coli K-12 strain AB2829. The nucleotide sequence of a 2.2-kb fragment encoding aroA predicted an open reading frame product 434 amino acids long that shows homology to other bacterial AroA proteins. Several strategies to inactivate aroA were unsuccessful. Gene replacement was finally achieved by constructing a replacement plasmid with aroA inactivated by insertion of a P. haemolytica ampicillin resistance fragment into a unique NdeI site in aroA. A hybrid plasmid was constructed by joining the aroA replacement plasmid with a 4.2-kb P. haemolytica plasmid which encodes streptomycin resistance. Following PhaI methylation, the replacement plasmid was introduced by electroporation into P. haemolytica NADC-D60, a plasmidless strain of serotype 1A. Allelic exchange between the replacement plasmid and the chromosome of P. haemolytica gave rise to an ampicillin-resistant mutant which grew on chemically defined P. haemolytica medium supplemented with aromatic amino acids but failed to grow on the same medium lacking tryptophan. Southern blot analysis confirmed that aroA of the mutant was inactivated and that the mutant was without a plasmid.     

副猪嗜血杆菌aroA基因鉴定及遗传进化分析

[目的]细菌aroA基因参与芳香族氨基酸的生物合成,被成功应用于细菌分类和基因失活致弱突变菌株的构建.副猪嗜血杆菌(Hps)是感染猪出现多发性浆膜炎和关节炎的一种病原细菌,鉴定该菌aroA全基因序列将有助于鉴定遗传进化关系和突变分析.[方法]利用PCR和细菌基因组步移技术鉴定Hps的aroA基因序列,进而对不同血清型菌株该基因序列进行鉴定,并与其它革兰氏阴性细菌进行比对和遗传进化分析.[结果]自Hps血清5型基因组DNA中获得包含完整aroA基因的3.7 kb基因片段,其中aroA基因全长1314 bp,编码产物长度437 aa,分子量大小47.9 kDa,该基因上游为磷酸烯醇式丙酮酸羧化酶基因.自本试验选择的Hps不同血清型菌株中均可扩增出包含完整aroA基因的1476 bp片段,且这些不同血清型菌株间核酸序列同源性在97.7%以上.Hps血清5型aroA基因序列与巴氏杆菌科其它成员核酸序列同源性为70.6%-78.9%,与E.coli和S.typhi-murium的同源性分别为66.4%和67.2%.[结论]本试验首次对Hps的15个血清型国际参考菌株及地方分离株aroA全基因序列进行了鉴定,序列比较结果显示aroA基因在革兰氏阴性细菌中具有较高的同源性.aroA基因鉴定对构建基因失活突变菌株以研究Hps生物学特性奠定了基础.     

Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant

The aroA gene from Pasteurella multocida was cloned by complementation of the Escherichia coli aroA mutant AB2829 with a DNA library constructed in pUC18. The nucleotide sequence of the P. multocida aroA gene indicated an open reading frame encoding a protein of 441 amino acids, which showed a high degree of homology with the amino acid sequences of various other bacterial AroA proteins. The cloned P. multocida aroA gene was inactivated by insertion of a kanamycin-resistance gene and reintroduced by allelic exchange into the chromosome of P. multocida using the suicide vector pJM703.1. The P. multocida aroA mutant was highly attenuated in a mouse model. Mice immunized intraperitoneally with two doses of live P. multocida aroA mutant were completely protected against a lethal parental strain challenge.     

Molecular and chemical characterization of the lipopolysaccharide O-antigen and its role in the virulence of Yersinia enterocolitica serotype O:8

The Y. enterocolitica O:8 (YeO8) O-antigen repeat units consist of five sugar residues: N-acetyl- d -galactosamine (GalNAc), d -galactose (Gal), d -mannose (Man), l -fucose (Fuc), and 6-deoxy- d -gulose (6d-Gul). The nucleotide sequence of the O-antigen gene cluster of the YeO8 strain 8081-c was determined. Altogether, 18 open reading frames (ORFs) were identified and shown to be essential for O-antigen biosynthesis. We previously characterized the 3'-end of the O-antigen gene cluster and identified four genes: two for GDP-Man biosynthesis, one for UDP-Gal biosynthesis, and one for O-antigen polymerase. Based on sequence similarity, Tn 5 -insertion phenotypes and chemical analysis, the 14 new genes were assigned the following functions: four genes are involved in the biosynthesis of CDP-6d-Gul and two in GDP-Fuc biosynthesis. Five gene products were assigned sugar transferase functions and one gene product was similar to Wzx, the O-antigen flippase. Two genes remained unassigned. By genetic complementation we also showed that YeO8 O-antigen biosynthesis was dependent on N-acetyl-glucosaminyl:undecaprenylphosphate transferase (GlcNAc transferase), the WecA (formerly known as Rfe) protein. Data obtained from chemical-composition analysis suggest that in addition to being GlcNAc transferase, WecA may also function as a GalNAc transferase. Using a restriction-deficient derivative of Y. enterocolitica O:8 strain 8081, a rough mutant, designated 8081-R2, was isolated. 8081-R2 was complemented in trans with a cloned O-antigen gene cluster restoring surface O-antigen expression. The virulence of the wild-type strain and that of the complemented strain were significantly higher (approx. 100-fold) than that of the rough mutant in an orally infected mouse model, showing that YeO8 O-antigen is a virulence factor.     

3-hydroxypyruvate substitutes for pyridoxine in serC mutants of Escherichia coli K-12.

Escherichia coli K-12 mutants with serC genotype required pyridoxine and serine for normal growth, as do E. coli B mutants of this type. Mutants of the K-12 strain, however, reverted easily to pyridoxine independence without regaining activity in the 3-phosphoserine oxoglutarate transaminase coded for by the serC gene. Both these revertants and the parental type synthesized pyridoxine in normal amounts when 3-hydroxypyruvate was used as a supplement, although neither of these mutants could use this compound to satisfy their serine requirement. Since serine alone was inadequate to provide the nutritional requirement of serC mutants, these mutants must have been unable to synthesize 3-hydroxypyruvate from serine. We suggest that 3-phosphoserine oxoglutarate transaminase in normal E. coli serves as a catalyst for transaminating small amounts of serine to 3-hydroxypyruvate, which is then used in pyridoxine biosynthesis. In serC mutants, this activity is blocked, and these mutants then show a double requirement for serine and pyridoxine.     

Genetic analysis of the O-specific lipopolysaccharide biosynthesis region (rfb) of Escherichia coli K-12 W3110: identification of genes that confer group 6 specificity to Shigella flexneri serotypes Y and 4a.

We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.     

Sequence, localization and function of the invasin protein of Yersinia enterocoiitica

The inv locus of Yersinia enterocolitica is sufficient to convert a non-invasive Escherichia coli K12 strain into a microorganism that is able to penetrate cultured mammalian cells. The nucleotide sequence of inv reveals an open reading frame corresponding to an 835-amino-acid protein that is homologous to the invasin protein from Yersinia pseudotuberculosis. A polyclonal antiserum elicited by a synthetic peptide corresponding to the C-terminal 88 amino acids of this open reading frame detected a unique 100 kD protein in cell lysates of Y. enterocolitica strain 8081 c and in an E. coli strain harbouring the cloned inv gene. This protein localized to the outer membranes of both microorganisms and was cleaved by low concentrations of extracellular trypsin. HEp-2 cells were shown to attach to surfaces coated with bacterial outer membranes containing invasin and this attachment was destroyed by treatment of the membranes with trypsin. Thus it appears that the invasin protein from Y. enterocolitica is able to mediate both attachment to and entry of cultured epithelial cells.     

Expression of Escherichia coli PhoE protein in avirulent Salmonella typhimurium aroA and galE strains

Abstract Escherichia coli K-12 PhoE protein is found to be normally expressed and incorporated into the outer membrane of two avirulent Salmonella typhimurium strains, G30 and SH aroA . A hybrid protein which contains an insertion of an antigenic epitope of VP1 protein of foot-and-mouth disease virus into the PhoE protein, was also normally assembled into the Salmonella outer membranes. In the case of the G30 stain, which carries a galE mutation, the inserted epitope is accessible to antibodies in intact cells. In contrast, the epitope is less accessible in the case of the SH aroA strain, probably due to the shielding effect of the O-antigen in this strain.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Acquisition of a sucrose utilization system in Escherichia coli K-12 derivatives and its application to industry.

An Escherichia coli strain, B-62, that was isolated from a clinical source and was epidemiologically unrelated to E. coli K-12 was the source of chromosomal DNA for a sucrose utilization system (Scr+) in the construction of a plasmid, pST621. The cloned insert of a gene encoding Scr+ in pST621 conferred a sucrose-positive phenotype onto transformed cells of E. coli K-12 derivatives. Sucrase activity of the transformants was as high as that which would correspond to a "gene dosage effect" of a vector plasmid pBR322, whereas the transformants' sucrose uptake activity was always lower than that of E. coli B-62. A region within an XhoI-SacI fragment (3.2 kb) of pBR322-glyA was replaced in the construction of another plasmid, pST5R7, by a fragment (about 2.6 kb) of pST622 containing the gene encoding Scr+. A genetically stable Scr+ derivative of E. coli K-12 was obtained by introducing the gene encoding Scr+ onto E. coli chromosome via homologous recombination between pST5R7 and the chromosome and subsequent plasmid segregation. The use of low-copy-number plasmid RP4 as a cloning vector was also effective for enhancing the stability of Scr+. Tryptophan producers E. coli SGIII1032S, in which the gene encoding Scr+ was cloned onto the chromosome, and E. coli SGIII1032, which carried Scr+ plasmid RP4.5R7, produced from 6% sucrose in shake flasks (33 degrees C, 96 h) 2.3 and 5.7 g of tryptophan per liter, respectively.     

Deletion of the RluD pseudouridine synthase promotes SsrA peptide tagging of ribosomal protein S7

RluD catalyses formation of three pseudouridine residues within helix 69 of the 50S ribosome subunit. Helix 69 makes important contacts with the decoding centre on the 30S subunit and deletion of rluD was recently shown to interfere with translation termination in Escherichia coli. Here, we show that deletion of rluD increases tmRNA activity on ribosomes undergoing release factor 2 (RF2)-mediated termination at UGA stop codons. Strikingly, tmRNA-mediated SsrA peptide tagging of two proteins, ribosomal protein S7 and LacI, was dramatically increased in ΔrluD cells. S7 tagging was due to a unique C-terminal peptide extension found in E. coli K-12 strains. Introduction of the rpsG gene (encoding S7) from an E. coli B strain abrogated S7 tagging in the ΔrluD background, and partially complemented the mutant's slow-growth phenotype. Additionally, exchange of the K-12 prfB gene (encoding RF2) with the B strain allele greatly reduced tagging in ΔrluD cells. In contrast to E. coli K-12 cells, deletion of rluD in an E. coli B strain resulted in no growth phenotype. These findings indicate that the originally observed rluD phenotypes result from synthetic interactions with rpsG and prfB alleles found within E. coli K-12 strains.     

Cloning of chromosomal beta-lactamase genes from Yersinia enterocolitica

Two beta-lactamase genes present in the chromosome of Yersinia enterocolitica have been cloned individually into the plasmid pACY184 and expressed in Escherichia coli. The gene for broad-spectrum beta-lactamase I ('A') was cloned from a strain belonging to the O:3 serotype, and the gene for (cephalosporinase) beta-lactamase II ('B') was cloned from a strain of the O:5b serotype. The properties of the beta-lactamases expressed in E. coli are similar to those previously described in Y. enterocolitica.     

Nonessential genes of phage phiYeO3-12 include genes involved in adaptation to growth on Yersinia enterocolitica serotype O:3

Bacteriophage phiYeO3-12 is a T7/T3-related lytic phage that naturally infects Yersinia enterocolitica serotype O:3 strains by using the lipopolysaccharide O polysaccharide (O antigen) as its receptor. The phage genome is a 39,600-bp-long linear, double-stranded DNA molecule that contains 58 genes. The roles of many of the genes are currently unknown. To identify nonessential genes, the isolated phage DNA was subjected to MuA transposase-catalyzed in vitro transposon insertion mutagenesis with a lacZ' gene-containing reporter transposon. Following electroporation into Escherichia coli DH10B and subsequent infection of E. coli JM109/pAY100, a strain that expresses the Y. enterocolitica O:3 O antigen on its surface, mutant phage clones were identified by their beta-galactosidase activity, manifested as a blue color on indicator plates. Transposon insertions were mapped in a total of 11 genes located in the early and middle regions of the phage genome. All of the mutants had efficiencies of plating (EOPs) and fitnesses identical to those of the wild-type phage when grown on E. coli JM109/pAY100. However, certain mutants exhibited altered phenotypes when grown on Y. enterocolitica O:3. Transposon insertions in genes 0.3 to 0.7 decreased the EOP on Y. enterocolitica O:3, while the corresponding deletions did not, suggesting that the low EOP was not caused by inactivation of the genes per se. Instead, it was shown that in these mutants the low EOP was due to the delayed expression of gene 1, coding for RNA polymerase. On the other hand, inactivation of gene 1.3 or 3.5 by either transposon insertion or deletion decreased phage fitness when grown on Y. enterocolitica. These results indicate that phiYeO3-12 has adapted to utilize Y. enterocolitica as its host and that these adaptations include the products of genes 1.3 and 3.5, DNA ligase and lysozyme, respectively.     

Cloning and expression of the Escherichia coli glgC gene from a mutant containing an ADPglucose pyrophosphorylase with altered allosteric properties.

A mutant strain of Escherichia coli K-12, designated 618, accumulates glycogen at a faster rate than wild-type strain 356. The mutation affects the ADPglucose pyrophosphorylase regulatory properties (N. Creuzat-Sigal, M. Latil-Damotte, J. Cattaneo, and J. Puig, p. 647-680, in R. Piras and H. G. Pontis, ed., Biochemistry of the Glycocide Linkage, 1972). The enzyme is less dependent on the activator, fructose 1,6 bis-phosphate for activity and is less sensitive to inhibition by the inhibitor, 5'-AMP. The structural gene, glgC, for this allosteric mutant enzyme was cloned into the bacterial plasmid pBR322 by inserting the chromosomal DNA at the PstI site. The glycogen biosynthetic genes were selected by cotransformation of the neighboring asd gene into an E. coli mutant also defective in branching enzyme (glgB) activity. Two recombinant plasmids, pEBL1 and pEBL3, that had PstI chromosomal DNA inserts containing glgC and glgB were isolated. Branching enzyme and ADPglucose pyrophosphorylase activities were increased 240- and 40-fold, respectively, in the asd glgB mutant, E. coli K-12 6281. The E. coli K-12 618 mutant glgC gene product was characterized after transformation of an E. coli B ADPglucose pyrophosphorylase mutant with the recombinant plasmid pEBL3. The kinetic properties of the cloned ADPglucose pyrophosphorylase were similar to those of the E. coli K-12 618 enzyme. The inserted DNA in pEBL1 was arranged in opposite orientation to that in pEBL3.     

Identification of the galE gene and a galE homolog and characterization of their roles in the biosynthesis of lipopolysaccharide in a serotype O:8 strain of Yersinia enterocolitica.

A clone that complements mutations in Yersinia enterocolitica lipopolysaccharide (LPS) core biosynthesis was isolated, and the DNA sequence of the clone was determined. Three complete open reading frames and one partial open reading frame were located on the cloned DNA fragment. The first, partial, open reading frame had homology to the rfbK gene. The remaining reading frames had homology to galE, rol, and gsk. Analysis of the galE homolog indicates that although it can complement an Escherichia coli galE mutant, its primary function in Y. enterocolitica is not in the production of UDP galactose but, instead, some other nucleotide sugar required for LPS biosynthesis. This gene has been renamed lse, for LPS sugar epimerase. The rol homolog has been demonstrated to have a role in Y. enterocolitica serotype 0:8 O-polysaccharide antigen chain length determination. An additional galE homolog has been identified in Y. enterocolitica by homology to the E. coli gene. The product of this gene has UDP galactose 4-epimerase activity in both E. coli and Y. enterocolitica. This gene is linked to the other genes of the galactose utilization pathway, similar to what is seen in other members of the family Enterobacteriaceae. Although Y. enterocolitica 0:8 strains are reported to have galactose as a constituent of LPS, a strain containing a mutation in this galE gene does not exhibit any LPS defects.