Cloning and Characterisation of the aroA and aroD Genes of Shigella dysenteriae Type 1

The aroA and aroD genes from Shigella dysenteriae type 1, encoding 5-enolpyruvylshikimate 3-phosphate synthase and 3-dehydroquinase, respectively, were cloned by polymerase chain reaction (PCR). Their nucleotide sequences were determined and predicted to code for 46 kDa and 27.5 kDa proteins, respectively. Protein expressed from these genes using the minicell system, corresponded to the size of the predicted protein products. The cloned genes were shown to be functional by complementation of Escherichia coli aroA and aroD^? mutants. The predicted amino acid sequences of the cloned aroA (427 amino acids) and aroD (252 amino acids) genes of S. dysenteriae type 1 were found to be highly homologous to the corresponding genes in other bacterial species, indicating the high conservation of these housekeeping genes. The use of the cloned aroA and aroD genes in the development of a vaccine strain against S. dysenteriae is discussed.     

The Mycobacterium tuberculosis shikimate pathway genes: Evolutionary relationship between biosynthetic and catabolic 3-dehydroquinases

Summary The Mycobacterium tuberculosis shikimate pathway genes designated aroB and aroQ encoding 3-dehydroquinate synthase and 3-dehydroquinase, respectively were isolated by molecular cloning and their nucleotide sequences determined. The deduced dehydroquinate synthase amino acid sequence from M. tuberculosis showed high similarity to those of equivalent enzymes from prokaryotes and filamentous fungi. Surprisingly, the deduced M. tuberculosis 3-dehydroquinase amino acid sequence showed no similarity to other characterised prokaryotic biosynthetic 3-dehydroquinases (bDHQases). A high degree of similarity was observed, however, to the fungal catabolic 3-dehydroquinases (cDHQases) which are active in the quinic acid utilisation pathway and are isozymes of the fungal bDHQases. This finding indicates a common ancestral origin for genes encoding the catabolic dehydroquinases of fungi and the biosynthetic dehydroquinases present in some prokaryotes. Deletion of genes encoding shikimate pathway enzymes represents a possible approach to generation of rationally attenuated strains of M. tuberculosis for use as live vaccines.     

Molecular cloning and characterization of the aroD gene encoding 3-dehydroquinase from Salmonella typhi

The aroD gene from Salmonella typhi, encoding 5-dehydroquinate hydrolyase (3-dehydroquinase), has been cloned into Escherichia coli and the DNA sequence determined. The aroD gene was isolated from a cosmid gene bank by complementation of an S. typhimurium aroD mutant. Analysis of the DNA sequence revealed the presence of an open reading frame capable of encoding a protein of 252 amino acids with a calculated Mr of 27706. Comparison of the deduced S. typhi 3-dehydroquinase protein sequence with that elucidated for E. coli revealed 69% homology. Alignment of the S. typhi sequence and equivalent Aspergillus nidulans and Saccharomyces cerevisiae sequences showed that homology was lower, at 24%, but still significant. Use of a minicell expression system demonstrated that a polyclonal antibody raised against E. coli 3-dehydroquinase cross-related with its S. typhi counterpart.     

Molecular organisation of the quinic acid utilization (QUT) gene cluster in Aspergillus nidulans

Summary The functional integrity of the QUTB gene (encoding quinate dehydrogenase) has been confirmed by transformation of a qutB mutant strain. The DNA sequence of the contiguous genes QUTD (quinate permease), QUTB and QUTG (function unknown) has been determined and analysed, together with that of QUTE (catabolic 3-dehydroquinase). The QUTB sequence shows significant homology with the shikimate dehydrogenase function of the complex AROM locus of Aspergillus nidulans, and with the QA-3 quinate dehydrogenase and QA-1S (repressor) genes of Neurospora crassa. The QUTD gene shows strong homology with the N. crassa QA-Y gene and QUTG with the QA-X gene. QUTD, QUTB, and QUTG, QUTE form two pairs of divergently transcribed genes, and conserved sequence motifs identified in the two common 5 non-coding regions show significant homology with UAS _GAL and UAS _QA sequences of the Saccharomyces cerevisiae and N. crassa Gal and QA systems. In addition, conserved 5 sequences homologous to the mammalian CAAT box are noted and a previously unreported conserved 22 nucleotide motif is presented.     

胸膜肺炎放线杆菌apxIC基因插入失活突变株构建及免疫原性分析

胸膜肺炎放线杆菌是引起猪传染性胸膜肺炎(APP)的呼吸道病原菌,其分泌的Apx毒素是最重要的毒力因子之一。为构建APP突变弱毒菌株,在apxIC基因下游XhoI酶切位点处插入氯霉素抗性基因(Chlr)制备转移载体,通过电转化导入APP血清10型参考菌株(D13039)进行同源重组,筛选获得apxIC基因插入突变菌株D13039C-Chl^r。该突变菌株特性鉴定结果表明其溶血活性完全丧失,可正常增殖和分泌ApxI毒素,连续10次传代后基因组中插入的Chl^r基因可稳定遗传,利用5个剂量(2×10⁸CFU~2×10⁶CFU)对每组3只小鼠腹腔攻毒结果显示突变菌株毒力较母源菌株降低至少100倍以上,将突变菌株作为弱毒活疫苗经滴鼻途径免疫仔猪后利用APP血清1型(4074)和血清10型(D13039)菌株攻毒进行免疫原性鉴定,结果显示血清1型攻毒后非免疫组4头仔猪全部死亡而免疫组4头中死亡2头,非免疫组肺损伤指数(34.4)显著高于免疫组(17.5),血清10型攻毒后非免疫组肺损伤指数(17.5)也高于免疫组(10.5),同时鼻拭子和肺组织样品的细菌重分离数及PCR检测阳性数非免疫组也明显高于免疫组,表明突变菌株作为弱毒活设疫苗对仔猪具有一定的交叉免疫保护力。该突变菌株的鉴定ApxI毒素活性及研制具有交叉保护活性的APP弱毒活疫苗奠定的基础。     

Cloning of histidine genes of Azospirillum brasilense: organization of the ABFH gene cluster and nucleotide sequence of the hisB gene

Summary A cluster of four Azospirillum brasilense histidine biosynthetic genes, hisA, hisB, hisF and hisH, was identified on a 4.5 kb DNA fragment and its organization studied by complementation analysis of Escherichia coli mutations and nucleotide sequence. The nucleotide sequence of a 1.3 kb fragment that complemented the E. coli hisB mutation was determined and an ORF of 624 nucleotides which can code for a protein of 207 amino acids was identified. A significant base sequence homology with the carboxyterminal moiety of the E. coli hisB gene (0.53) and the Saccharomyces cerevisiae HIS3 gene (0.44), coding for an imidazole glycerolphosphate dehydratase activity was found. The amino acid sequence and composition, the hydropathic profile and the predicted secondary structures of the yeast, E. coli and A. brasilense proteins were compared. The significance of the data presented is discussed.Abbreviations IGP imidazole glycerolphosphate - HP histidinolphosphate     

Predicting genetic traits and epitope analysis of <Emphasis Type="Italic">apxIVA</Emphasis> in <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis>

Actinobacillus pleuropneumoniae causes a severe hemorrhagic pneumonia in pigs. Fifteen serotypes of A. pleuropneumoniae express four different Apx toxins that belong to the pore-forming repeats-in-toxin (RTX) group of toxins. ApxIV, which is conserved and up-regulated in vivo, could be an excellent candidate for the development of a protective cross-serotype immunity vaccine, and could aid in the differential diagnosis of diseases caused by A. pleuropneumoniae. We identified and sequenced apxIVA from A. pleuropneumoniae serotype 2 isolated in Korea (Kor-ApxIVA). The Kor-ApxIVA was closely related to Switzerland (AF021919), China (CP000687), and China (GQ332268), showing 98.6%, 98.4%, and 97.2% amino acid homology, respectively. The level of amino acid homology, however, was higher than the nucleotide homology. The structural characteristics of ApxIVA showed RTX proteins, including N-terminal hydrophobic domains, signature sequences for potential acylation sites, and repeated glycine-rich nonapeptides in the C-terminal region of the protein. Thirty glycine-rich nonapeptides with the consensus sequence, L/V-X-G-G-X-G-N/D-D-X, were found in the C-terminus of the Kor-ApxIVA. In addition, the Kor-ApxIVA was predicted for the linear B-cell epitopes and conserved domains with determined peptide sequences. This genetic analysis of the Kor-ApxIVA might be an important foundation for future biological and functional research on ApxIVA.     

malT knockout mutation invokes a stringent type gene-expression profile in Actinobacillus pleuropneumoniae in bronchoalveolar fluid

Background  

Actinobacillus pleuropneumoniae causes contagious pleuropneumonia, an economically important disease of commercially reared pigs throughout the world. To cause this disease, A. pleuropneumoniae must rapidly overcome porcine pulmonary innate immune defenses. Since bronchoalveolar fluid (BALF) contains many of the innate immune and other components found in the lungs, we examined the gene expression of a virulent serovar 1 strain of A. pleuropneumoniae after exposure to concentrated BALF for 30 min.     

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.     

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

Summary The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leu4 and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicellproducing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known -D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Cloning and Characterization of Xylanase A from the Strain <Emphasis Type="Italic">Bacillus</Emphasis> sp. BP-7: Comparison with Alkaline pI-Low Molecular Weight Xylanases of Family 11

The xynA gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-7 has been cloned and expressed in Escherichia coli. Recombinant xylanase A showed high activity on xylans from hardwoods and cereals, and exhibited maximum activity at pH 6 and 60°C. The enzyme remained stable after incubation at 50°C and pH 7 for 3 h, and it was strongly inhibited by Mn²⁺, Fe³⁺, Pb²⁺, and Hg²⁺. Analysis of xylanase A in zymograms showed an apparent molecular size of 24 kDa and a pI of above 9. The amino acid sequence of xylanase A, as deduced from xynA gene, shows homology to alkaline pI-low molecular weight xylanases of family 11 such as XynA from Bacillus subtilis. Analysis of codon usage in xynA from Bacillus sp. BP-7 shows that the G+C content at the first and second codon positions is notably different from the mean values found for glycosyl hydrolase genes from Bacillus subtilis.     

Evidence for Gene Duplication and Allelic Codominance (not Hierarchical Dominance) at the Mating‐Type Locus of the Ciliate,Euplotes crassus

The high‐multiple mating system of Euplotes crassus is known to be controlled by multiple alleles segregating at a single locus and manifesting relationships of hierarchical dominance, so that heterozygous cells would produce a single mating‐type substance (pheromone). In strain L‐2D, now known to be homozygous at the mating‐type locus, we previously identified two pheromones (Ec‐α and Ec‐1) characterized by significant variations in their amino acid sequences and structure of their macronuclear coding genes. In this study, pheromones and macronuclear coding genes have been analyzed in strain POR‐73 characterized by a heterozygous genotype and strong mating compatibility with L‐2D strain. It was found that POR‐73 cells contain three distinct pheromone coding genes and, accordingly, secrete three distinct pheromones. One pheromone revealed structural identity in amino acid sequence and macronuclear coding gene to the Ec‐α pheromone of L‐2D cells. The other two pheromones were shown to be new and were designated Ec‐2 and Ec‐3 to denote their structural homology with the Ec‐1 pheromone of L‐2D cells. We interpreted these results as evidence of a phenomenon of gene duplication at the E. crassus mating‐type locus, and lack of hierarchical dominance in the expression of the macronuclear pheromone genes in cells with heterozygous genotypes.     

A T4 DNA fragment containing genes for the baseplate central plug: Endonuclease restriction, gene expression and cell wall changes

Summary A fragment of Escherichia coli bacteriophage T4D DNA, containing 6.1 Kbp which included the six genes (genes 25, 26, 51, 27, 28 and 29) coding for the tail baseplate central plug has been partially characterized. This DNA fragment was obtained originally by Wilson et al. (1977) by the action of the restriction enzyme EcoRI on a modified form of T4 DNA and was inserted in the pBR322 plasmid and then incorporated into an E. coli K12 strain called RRI. This plasmid containing the phage DNA fragment has now been reisolated and screened for cleavage sites for various restriction endonucleases. Restriction enzymes Bgl 11 and Xbal each attacked one restriction site and the enzyme Hpa 1 attacked two restriction sites on this fragment. The combined digestion of the hybrid plasmid containing the T4 EcoRI DNA fragment conjugated to the pBR322 plasmid with one of these enzymes plus Bam H1 restriction enzyme resulted in the localization of the restriction site for Bgl 11, Xba 1 and Hpa 1. Escherichia coli strain B cells were transformed with this hybrid plasmid and found to have some unexpected properties. E. coli B cells, which are normally restrictive for T4 amber mutants and for T4 temperature sensitive mutants (at 44°) after transformation, were permissive for 25am, 26am and 26^Ts, 51am, and 51^Ts, 27^Ts, and 28^Ts T4 mutants. Extracts from the transformed E. coli cells were found in complementation experiments to contain the gene 29 product, as well as the gene 26 product, the gene 51 product, and the gene 27 product. The complementation experiments and the permissiveness of the transformed E. coli B cells to the various conditional lethal mutants clearly showed that the six T4 genes were producing all six gene products in these transformed cells. However, these cells were not permissive for T4 amber mutants in genes 27, 28, and 29. The transformed E. coli B cells, as compared to untransformed cells, were found to have altered outer cell walls which made them highly labile to osmotic shock and to an increased rate of killing by wild type T4 and all T4 amber mutants except for T4 am29. The change in cell walls of the transformed cells has been found to be due to the T4 baseplate genes on the hybrid plasmid, since E. coli B transformed by the pBR322 plasmid alone does not show the increase in osmotic sensitivity.     

Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

The gene fimU affects expression of Salmonella typhimurium type 1 fimbriae and is related to the Escherichia coli tRNA gene argU

The gene fimU, located on a recombinant plasmid carrying the Salmonella typhimurium type 1 fimbrial gene cluster is closely related to the Escherichia coli tRNA gene argU. The fimU gene complements an E. coli argU mutant that is a P2 lysogen, thereby allowing the phage P4 to grow in this strain but preventing the growth of phage lambda. In addition, fimU was shown to be involved in fimbrial expression since transformants of the E. coli argU mutant could produce fimbriae only in the presence of fimU but not in its absence, whereas in an E. coli argU ⁺ strain fimbriation did not require the fimU gene.     

In vivo overproduction of the pentafunctional arom polypeptide in Aspergillus nidulans affects metabolic flux in the quinate pathway

Summary The shikimate pathway and the quinic acid utilisation (QUT) pathway of Aspergillus nidulans and other fungi share the two common metabolic intermediates, 3-dehydroquinic acid (DHQ) and dehydroshikimic acid (DHS), which are interconverted by two isoenzymes, catabolic 3-dehydroquinase, (cDHQase) and biosynthetic dehydroquinase (bDHQase). bDHQase is one of five consecutive enzymatic activities associated with the pentafunctional arom protein encoded by the complex AROM locus, whereas cDHQase is encoded by the single-function QUTE gene, one of seven genes comprising the QUT gene cluster in A. nidulans, which is required for the catabolism of quinate to protocatechuate. We addressed the question of how much (if any) leakage there is of the two common substrates between the two pathways, by increasing the concentration of the arom protein in vivo by means of recombinant DNA technology. We demonstrated that constitutive overproduction of the arom protein by 12-fold in the presence of quinate inhibits germination of conidiospores, but showed that 12-fold quinate-inducible overproduction of arom protein does not have this effect. In addition we showed that a qutE mutant (lacking cDHQase) can grow with quinic acid as sole carbon source when the arom protein is overproduced fivefold. The data are most simply interpreted as simple competition for common substrates by the enzymes of the two pathways and demonstrate that any channelling function of the arom protein in vivo is relatively leaky.