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PubMed1992

R-plasmid-encoded adhesive factor in Klebsiella pneumoniae strains responsible for human nosocomial infections.

Klebsiella pneumoniae strains involved in hospital outbreaks of nosocomial infections, such as suppurative lesions, bacteremia, and septicemia, were resistant to multiple antibiotics including broad-spectrum cephalosporins. Epidemiologic investigations revealed that the reservoir for these K. pneumoniae strains was the gastrointestinal tracts of the patients. The study of the adherence ability of the strains reported here showed that these bacteria adhered to the microvilli of the Caco-2 cell line. This adhesion was mediated by a nonfimbrial protein with a molecular mass of 29,000 Da designated CF29K. Pretreatment of bacteria with antibodies raised against CF29K or Caco-2 cells with purified CF29K prevented the adhesion of K. pneumoniae strains to Caco-2 cells. CF29K immunologically cross-reacted with the CS31A surface protein of Escherichia coli strains involved in septicemia in calves. Genes encoding CF29K were located on a high-molecular-weight conjugative R plasmid, which transferred to E. coli K-12. Transconjugants expressed a large amount of CF29K protein and adhered to the brush border of Caco-2 cells. These findings show that K. pneumoniae strains were able to colonize the human intestinal tract through a plasmid-encoded 29,000-Da surface protein. Hybridization experiments indicated that the gene encoding resistance to broad-spectrum cephalosporins by the production of CAZ-1 enzyme and the gene encoding the adhesive property to intestinal cells were both located on a 20- to 22-kb EcoRI restriction DNA fragment. Genes encoding aerobactin and the ferric aerobactin receptor were also found on this R plasmid.

Bacterial Adhesion↗

PubMed1992

Cloning of a gene (zot) encoding a new toxin produced by Vibrio cholerae.

Live oral candidate cholera vaccines have previously been constructed by deletion of Vibrio cholerae sequences encoding the enzymatically active A subunit of the cholera toxin. However, volunteer studies have shown that these non-cholera toxin-producing strains still provoke mild to moderate diarrhea in some individuals. We recently reported the identification of a second toxin produced by V. cholerae which may be responsible for this residual diarrhea (A. Fasano, B. Baudry, D. W. Pumplin, S. S. Wasserman, B. D. Tall, J. M. Ketley, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 88:5242-5246, 1991). This new toxigenic factor increases the permeability of rabbit ileal mucosa by affecting the structure of the intercellular tight junctions (zonula occludens). We now report the identification and cloning of the gene encoding this new toxin. This gene, named zot (for zonula occludens toxin), consists of a 1.3-kb open reading frame which could potentially encode a 44.8-kDa polypeptide. The location of the zot gene encoding the new toxin is immediately upstream of the ctx operon encoding cholera toxin.

Amino Acid Sequence↗

PubMed1992

Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis.

A group of high-molecular-weight surface-exposed proteins of nontypeable Haemophilus influenzae are major targets of human serum antibody (S. J. Barenkamp and F. F. Bodor, Pediatr. Infect. Dis. J. 9:333-337, 1990). To further characterize these proteins, we cloned and sequenced genes encoding two related high-molecular-weight proteins from a prototype nontypeable Haemophilus strain. The gene encoding a 120-kDa Haemophilus protein consisted of a 4.4-kbp open reading frame, and the gene encoding a 125-kDa protein consisted of a 4.6-kbp open reading frame. The first 1,259 bp of the two genes were identical. Thereafter, the sequences began to diverge, but overall they were 80% identical, and the derived amino acid sequences showed 70% identity. A protein sequence homology search demonstrated similarity between the derived amino acid sequences of both cloned genes and the derived amino acid sequence of the gene encoding filamentous hemagglutinin, a surface protein produced by the gram-negative pathogen Bordetella pertussis. Antiserum raised against a recombinant protein encoded by the 4.6-kbp open reading frame recognized both the 120- and the 125-kDa proteins in the prototype strain as well as antigenically related high-molecular-weight proteins in 75% of a collection of 125 epidemiologically unrelated nontypeable H. influenzae strains. The antiserum directed against the recombinant protein also recognized purified filamentous hemagglutinin. A murine monoclonal antibody to filamentous hemagglutinin recognized both the 120-kDa and the 125-kDa protein in the prototype strain as well as proteins identical to those recognized by the recombinant-protein antiserum in 35% of the nontypeable H. influenzae strain collection. Thus, we have identified and partially characterized a group of highly immunogenic surface-exposed proteins of nontypeable H. influenzae which are related to the filamentous hemagglutinin of B. pertussis.

Adhesins, Bacterial↗

PubMed1993

Genetic analysis of the gene cluster encoding nonfimbrial adhesin I from an Escherichia coli uropathogen.

The chromosomally encoded nonfimbrial adhesion I (NFA-I) from Escherichia coli urinary tract isolate 827 (O83:K1:H4) mediates agglutination of human erythrocytes. Subclones were constructed from an NFA-I-expressing recombinant E. coli K-12 clone, derived from a genomic library of E. coli 827. Minicell analysis and nucleotide sequencing revealed that proteins of 30.5, 9, 80, 15, and 19 kDa encoded on a stretch of approximately 6 kb are involved in the expression of NFA-I. NFA-I exhibits a polymeric structure, which disintegrates with elevated temperature into a 19-kDa monomer but with some relatively stable dimers. By using gold-conjugated monoclonal antibodies directed against NFA-I in electron microscopy, the adhesin could be localized on the outer surface of the recombinant E. coli K-12 bacteria. The nucleotide sequence of the nfaA gene encoding the monomeric structural subunit of the adhesin was determined. An open reading frame of 184 amino acids encoding the NfaA precursor, which is processed to the mature protein, was found; it consisted of 156 amino acids with a calculated molecular weight of 16,000. Peptide sequencing of the NFA-I subunit protein confirmed that this open reading frame corresponds to the NfaA coding locus. Furthermore, the nucleotide sequence of the open reading frame termed NfaE, located at the proximal part of the DNA stretch responsible for NFA-I expression, was elaborated. NfaE consists of 247 amino acids, including a presumptive 29-amino-acid signal peptide, leading to a molecular weight of 24,000 for the mature protein. The nfaE sequence shares homology with the 27-kDa CS3 protein, which is involved in the assembly of CS3 fibrillae, and might encode the 30.5-kDa protein, detected in minicells.

Adhesins, Escherichia coli↗

PubMed1999

Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island.

Upon entry into the host, Salmonella enterica strains are presumed to encounter an iron-restricted environment. Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment. We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island of Salmonella typhimurium. The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of the S. typhimurium chromosome. This locus, designated sit, consists of four open reading frames which encode polypeptides with extensive homology to the yfe ABC iron transport system of Yersinia pestis, as well as other ABC transporters. The sitA gene encodes a putative periplasmic binding protein, sitB encodes an ATP-binding protein, and sitC and sitD encode two putative permeases (integral membrane proteins). This operon is capable of complementing the growth defect of the enterobactin-deficient Escherichia coli strain SAB11 in iron-restricted minimal medium. Transcription of the sit operon is repressed under iron-rich growth conditions in a fur-dependent manner. Introduction of a sitBCD deletion into wild-type S. typhimurium resulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype. These results further support the existence of redundant iron uptake systems in S. enterica.

ATP-Binding Cassette Transporters↗

PubMed2001

Induction of cell-mediated immunity against Mycobacterium tuberculosis using DNA vaccines encoding cytotoxic and helper T-cell epitopes of the 38-kilodalton protein.

Cell-mediated immune responses are crucial in the protection against tuberculosis. In this study, we constructed DNA vaccines encoding cytotoxic T lymphocytes (CTL) and T helper cell (Th) epitopes of the 38-kDa lipoglycoprotein of Mycobacterium tuberculosis and analyzed and compared their immunogenicities with that of pXJ38, a DNA vaccine encoding the entire 38-kDa protein (X. Zhu, N. Venkataprasad, H. S. Thangaraj, M. Hill, M. Singh, J. Ivanyi, and H. M. Vordermeier, J. Immunol. 158:5921-5926, 1997). Plasmid DNAs encoding a CTL epitope, P3 (pP3), a Th epitope (vTh), or both the Th and the P3 epitopes (pThP3) were prepared and tested in C57BL6/J (H-2(b)) mice. Our results confirmed that DNA immunization with pXJ38 induces strong CD8(+) CTL and Th1 responses (high gamma interferon [IFN-gamma], low interleukin-4 [IL-4]). Coadministration of plasmid DNAs encoding a Th epitope with those encoding a CTL epitope (vTh+pP3) elicited both antigen-specific CD8(+) CTL and Th1 responses. High levels of IFN-gamma were secreted by spleen cells from all plasmid DNA-vaccinated mice after in vitro stimulation with the recombinant 38-kDa protein. Small or undetectable amounts of IL-4 were observed, which indicates the induction of a Th1-like response. Multiple-epitope vaccination by vTh+pP3 or pThP3 resulted in a broader Th1 response to peptide or epitopes than the single-epitope plasmid DNAs. Antigen-specific immunoglobulin G2a was only detected in sera from mice immunized with the plasmid pXJ38, and not in mice immunized with the epitope-based DNA vaccines. Thus, the absence of an antibody response after immunization with epitope plasmid DNAs and their ability to trigger only a specific cellular immune response may prove to be important advantages for a vaccine against tuberculosis.

Animals↗

PubMed2004

Regulators encoded in the Escherichia coli type III secretion system 2 gene cluster influence expression of genes within the locus for enterocyte effacement in enterohemorrhagic E. coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 subverts host cells through a type III secretion system encoded by the locus for enterocyte effacement (LEE). Genome sequencing of this pathotype revealed the existence of a gene cluster encoding components of a second cryptic type III secretion system, E. coli type III secretion system 2 (ETT2). Recently, we showed that the ETT2 gene cluster is present in whole or in part in the majority of E. coli strains but is unable to encode a functional secretion system in most strains, including EHEC O157:H7. However, here we show that mutational inhibition of two regulatory genes (ECs3720 or etrA and ECs3734 or eivF) from the ETT2 cluster in EHEC O157:H7 leads to greatly increased secretion of proteins encoded by the LEE and to increased adhesion to human intestinal cells. Studies in which transcriptional fusions and microarrays were used indicated that EtrA and EivF exert profound negative effects on gene transcription within the LEE. Consistent with these observations, expression of these regulators in an EHEC O26:H- strain led to suppression of protein secretion under LEE-inducing conditions. These findings provide fresh examples of the influence of mobile genetic elements on regulation of the LEE and of cross talk between type III secretion system gene clusters. In addition, they provide a cautionary tale because they show that the effects of regulatory genes can outlive widespread decay of other genes in a functionally coherent gene cluster, a phenomenon that we have named the "Cheshire cat effect." It also seems likely that variations in the ETT2 regulator repertoire might account for strain-to-strain variation in secretion of LEE-encoded proteins.

Bacterial Adhesion↗

PubMed2005

The locus of enterocyte effacement-encoded effector proteins all promote enterohemorrhagic Escherichia coli pathogenicity in infant rabbits.

The genes encoding the enterohemorrhagic Escherichia coli (EHEC) type III secretion system (TTSS) and five effector proteins secreted by the TTSS are located on the locus of enterocyte effacement (LEE) pathogenicity island. Deletion of tir, which encodes one of these effector proteins, results in a profound reduction (approximately 10,000-fold) in EHEC colonization of the infant rabbit intestine, but the in vivo phenotypes of other LEE genes are unknown. Here, we constructed in-frame deletions in escN, the putative ATPase component of the TTSS, and the genes encoding the four other LEE-encoded effector proteins, EspH, Map, EspF, and EspG, to investigate the contributions of the TTSS and the translocated effector proteins to EHEC pathogenicity in infant rabbits. We found that the TTSS is required for EHEC colonization and attaching and effacing (A/E) lesion formation in the rabbit intestine. Deletion of escN reduced EHEC recovery from the rabbit intestine by approximately 10,000-fold. Although EspH, Map, EspF, and EspG were not required for A/E lesion formation in the rabbit intestine or in HeLa cells, these effector proteins promote EHEC colonization. Colonization by the espH and espF mutants was reduced throughout the intestine. In contrast, colonization by the map and espG mutants was reduced only in the small intestine, indicating that Map and EspG have organ-specific effects. EspF appears to down-regulate the host response to EHEC, since we observed increased accumulation of polymorphonuclear leukocytes in the colonic mucosa of rabbits infected with the EHEC espF mutant. Thus, all the known LEE-encoded effector proteins influence EHEC pathogenicity.

Animals↗

PubMed2005

Expression of genes encoding innate host defense molecules in normal human monocytes in response to Candida albicans.

Little is known about the regulation and coordinated expression of genes involved in the innate host response to Candida albicans. We therefore examined the kinetic profile of gene expression of innate host defense molecules in normal human monocytes infected with C. albicans using microarray technology. Freshly isolated peripheral blood monocytes from five healthy donors were incubated with C. albicans for 0 to 18 h in parallel with time-matched uninfected control cells. RNA from monocytes was extracted and amplified for microarray analysis, using a 42,421-gene cDNA chip. Expression of genes encoding proinflammatory cytokines, including tumor necrosis factor alpha, interleukin 1 (IL-1), IL-6, and leukemia inhibitory factor, was markedly enhanced during the first 6 h and coincided with an increase in phagocytosis. Expression of these genes returned to near baseline by 18 h. Genes encoding chemokines, including IL-8; macrophage inflammatory proteins 1, 3, and 4; and monocyte chemoattractant protein 1, also were strongly up-regulated, with peak expression at 4 to 6 h, as were genes encoding chemokine receptors CCR1, CCR5, CCR7, and CXCR5. Expression of genes whose products may protect monocyte viability, such as BCL2-related protein, metallothioneins, CD71, and SOCS3, was up-regulated at 4 to 6 h and remained elevated throughout the 18-h time course. On the other hand, expression of genes encoding T-cell-regulatory molecules (e.g., IL-12, gamma interferon, and transforming growth factor beta) was not significantly affected during the 18-h incubation. Moreover, genes encoding IL-15, the IL-13 receptor (IL-13Ra1), and CD14 were suppressed during the 18-h exposure to C. albicans. Thus, C. albicans is a potent inducer of a dynamic cascade of expression of genes whose products are related to the recruitment, activation, and protection of neutrophils and monocytes.

Antigens, CD↗

PubMed2006

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase.

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.

Amino Acid Sequence↗

PubMed2006

A gene cluster involved in degradation of substituted salicylates via ortho cleavage in Pseudomonas sp. strain MT1 encodes enzymes specifically adapted for transformation of 4-methylcatechol and 3-methylmuconate.

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

Base Sequence↗

PubMed1986

Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes.

A gene cluster encoding biphenyl- and chlorobiphenyl-degrading enzymes was cloned from a soil pseudomonad into Pseudomonas aeruginosa PAO1161. Chromosomal DNA from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes KF707 was digested with restriction endonuclease XhoI and cloned into the unique XhoI site of broad-host-range plasmid pKF330. Of 8,000 transformants tested, only 1, containing the chimeric plasmid pMFB1, rendered the host cell able to convert biphenyls and chlorobiphenyls to ring meta cleavage compounds via dihydrodiols and dihydroxy compounds. The chimeric plasmid contained a 7.9-kilobase XhoI insert. Subcloning experiments revealed that the genes bphA (encoding biphenyl dioxygenase), bphB (encoding dihydrodiol dehydrogenase), and bphC (encoding 2,3-dihydroxybiphenyl dioxygenase) were coded for by the 7.9-kilobase fragment. The gene order was bphA-bphB-bphC. The hydrolase activity, which converted the intermediate meta cleavage compounds to the final product, chlorobenzoic acids, and was encoded by a putative bphD gene, was missing from the cloned 7.9-kilobase fragment.

Alcohol Oxidoreductases↗

PubMed1988

Structure and comparative analysis of the genes encoding component C of methyl coenzyme M reductase in the extremely thermophilic archaebacterium Methanothermus fervidus.

A 6-kilobase-pair (kbp) region of the genome of the extremely thermophilic arachaebacterium Methanothermus fervidus which encodes the alpha, beta, and gamma subunit polypeptides of component C of methyl coenzyme M reductase was cloned and sequenced. Genes encoding the beta (mcrB) and gamma (mcrG) subunits were separated by two open reading frames (designated mcrC and mcrD) which encode unknown gene products. The M. fervidus genes were preceded by ribosome-binding sites, separated by short A + T-rich intergenic regions, contained unexpectedly few NNC codons, and exhibited inflexible codon usage at some locations. Sites of transcription initiation and termination flanking the mcrBDCGA cluster of genes in M. fervidus were identified. The sequences of the genes, the encoded polypeptides, and transcription regulatory signals in M. fervidus were compared with the functionally equivalent sequences from two mesophilic methanogens (Methanococcus vannielii and Methanosarcina barkeri) and from a moderate thermophile (Methanobacterium thermoautotrophicum Marburg). The amino acid sequences of the polypeptides encoded by the mcrBCGA genes in the two thermophiles were approximately 80% identical, whereas all other pairs of these gene products contained between 50 and 60% identical amino acid residues. The mcrD gene products have diverged more than the products of the other mcr genes. Identification of highly conserved regions within mcrA and mcrB suggested oligonucleotide sequences which might be developed as hybridization probes which could be used for identifying and quantifying all methanogens.

Amino Acid Sequence↗

PubMed1990

Primary sequence of the Escherichia coli fadBA operon, encoding the fatty acid-oxidizing multienzyme complex, indicates a high degree of homology to eucaryotic enzymes.

In Escherichia coli at least five enzyme activities required for the beta-oxidation of fatty acids are associated with a multienzyme complex composed of two subunits in alpha 2 beta 2 conformation (A. Pramanik et al., J. Bacteriol. 137:469-473, 1979). In the present work, the DNA sequence of the genes encoding these two subunits, fadB and fadA, has been determined. The direction of transcription was from fadB to fadA rather than from fadA to fadB, as suggested previously (S. K. Spratt et al., J. Bacteriol. 158:535-542, 1984). Only 10 nucleotides separated the coding sequences for the two peptides, confirming the suggestion that these genes form an operon. The peptides encoded by fadB and fadA were 729 amino acids and 387 amino acids, respectively, in length. The larger and smaller peptides had predicted molecular masses of 79,678 and 40,876 Da, respectively. Recently, the sequence of the fadA gene was published in a separate report (Yang et al., J. Biol. Chem. 265:10424-10429, 1990). In this work, most of the DNA sequence for fadA was confirmed, and 10 errors were corrected. Three of these nucleotide changes resulted in five amino acid residue changes predicted in the carboxy terminus of the fadA-encoded peptide. By comparison to other peptide sequences, the alpha subunit encoded within fadB had 31% perfect identity with the rat peroxisomal enoyl-coenzyme A:hydratase-3-hydroxyacyl-coenzyme A dehydrogenase trifunctional enzyme over the entire length of the two peptides. In agreement with the work of Yang et al., the beta subunit encoded within fadA had 35 to 45% perfect identity with five thiolase genes from different eucaryotic sources over the entire length of the peptide.

Amino Acid Sequence↗

PubMed1991

Cloning and characterization of the Salmonella typhimurium ada gene, which encodes O6-methylguanine-DNA methyltransferase.

The ada gene of Escherichia coli encodes O6-methylguanine-DNA methyltransferase, which serves as a positive regulator of the adaptive response to alkylating agents and as a DNA repair enzyme. The gene which can make an ada-deficient strain of E. coli resistant to the cell-killing and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) has been cloned from Salmonella typhimurium TA1538. DNA sequence analysis indicated that the gene potentially encoded a protein with a calculated molecular weight of 39,217. Since the nucleotide sequence of the cloned gene shows 70% similarity to the ada gene of E. coli and there is an ada box-like sequence (5'-GAATTAAAACGCA-3') in the promoter region, we tentatively refer to this cloned DNA as the adaST gene. The gene encodes Cys-68 and Cys-320, which are potential acceptor sites for the methyl group from the damaged DNA. The multicopy plasmid carrying the adaST gene significantly reduced the frequency of mutation induced by MNNG both in E. coli and in S. typhimurium. The AdaST protein encoded by the plasmid increased expression of the ada'-lacZ chromosome fusion about 5-fold when an E. coli strain carrying both the fusion operon and the plasmid was exposed to a low concentration of MNNG, whereas the E. coli Ada protein encoded by a low-copy-number plasmid increased it about 40-fold under the same conditions. The low ability of AdaST to function as a positive regulator could account for the apparent lack of an adaptive response to alkylation damage in S. typhimurium.

Amino Acid Sequence↗

PubMed1991

A gene encoding lysine 6-aminotransferase, which forms the beta-lactam precursor alpha-aminoadipic acid, is located in the cluster of cephamycin biosynthetic genes in Nocardia lactamdurans.

A gene (lat) encoding lysine 6-aminotransferase was found upstream of the pcbAB (encoding alpha-aminoadipylcysteinyl-valine synthetase) and pcbC (encoding isopenicillin N synthase) genes in the cluster of early cephamycin biosynthetic genes in Nocardia lactamdurans. The lat gene was separated by a small intergenic region of 64 bp from the 5' end of the pcbAB gene. The lat gene contained an open reading frame of 1,353 nucleotides (71.4% G + C) encoding a protein of 450 amino acids with a deduced molecular mass of 48,811 Da. Expression of DNA fragments carrying the lat gene in Streptomyces lividans led to a high lysine 6-aminotransferase activity which was absent from untransformed S. lividans. The enzyme was partially purified from S. lividans(pULBS8) and showed a molecular mass of 52,800 Da as calculated by Sephadex gel filtration and polyacrylamide gel electrophoresis. DNA sequences which hybridized strongly with the lat gene of N. lactamdurans were found in four cephamycin-producing Streptomyces species but not in four other actinomycetes which are not known to produce beta-lactams, suggesting that the gene is specific for beta-lactam biosynthesis and is not involved in general lysine catabolism. The protein encoded by the lat gene showed similarity to ornithine-5-aminotransferases and N-acetylornithine-5-aminotransferases and contained a pyridoxal phosphate-binding consensus amino acid sequence around Lys-300 of the protein. The evolutionary implications of the lat gene as a true beta-lactam biosynthetic gene are discussed.

2-Aminoadipic Acid↗

PubMed1992

The cefG gene of Cephalosporium acremonium is linked to the cefEF gene and encodes a deacetylcephalosporin C acetyltransferase closely related to homoserine O-acetyltransferase.

The gene (cefG) encoding the acetyl coenzyme A:deacetylcephalosporin C acetyltransferase of Cephalosporium acremonium (synonym Acremonium chrysogenum) C10 has been cloned. It contains two introns and encodes a protein of 444 amino acids with an M(r) of 49,269 that correlates well with the M(r) deduced by gel filtration. The cefG gene is linked to the cefEF gene (encoding the bifunctional deacetoxycephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the cefEF gene. Two transcripts of 1.2 and 1.4 kb were found in C. acremonium that correspond to the cefEF and cefG genes, respectively; the degree of expression of the cefG gene was clearly lower than that of the cefEF gene in 48-h cultures. The cloned cefG complemented the deficiency of deacetylcephalosporin acetyltransferase in the nonproducer mutant C. acremonium ATCC 20371 and restored cephalosporin biosynthesis in this strain. Heterologous expression of the cefG genes took place in Penicillium chrysogenum. The deacetylcephalosporin acetyltransferase showed a much higher degree of homology with the O-acetylhomoserine acetyltransferases of Saccharomyces cerevisiae and Ascobolus immersus than with other O-acetyltransferases. The cefEF-cefG cluster of genes encodes the enzymes that carry out the three late steps of the cephalosporin biosynthetic pathway and is not linked to the pcbAB-pcbC gene cluster that encodes the first two steps of the pathway.

Acetyltransferases↗

PubMed1993

Identification, expression, and DNA sequence of the GDP-mannose biosynthesis genes encoded by the O7 rfb gene cluster of strain VW187 (Escherichia coli O7:K1).

The O7-specific lipopolysaccharide (LPS) in strains of Escherichia coli consists of a repeating unit made of galactose, mannose, rhamnose, 4-acetamido-2,6-dideoxyglucose, and N-acetylglucosamine. We have recently cloned and characterized genetically the O7-specific LPS biosynthesis region (rfbEcO7) of the E. coli O7:K1 strain VW187 (C. L. Marolda, J. Welsh, L. Dafoe, and M. A. Valvano, J. Bacteriol. 172:3590-3599, 1990). In this study, we localized the gnd gene encoding gluconate-6-phosphate dehydrogenase at one end of the rfbEcO7 gene cluster and sequenced that end of the cluster. Three open reading frames (ORF) encoding polypeptides of 275, 464, and 453 amino acids were identified upstream of gndEcO7, all transcribed toward the gnd gene. ORF275 had 45% similarity at the protein level with ORF16.5, which occupies a similar position in the Salmonella enterica LT2 rfb region, and presumably encodes a nucleotide sugar transferase. The polypeptides encoded by ORFs 464 and 453 were expressed under the control of the ptac promoter and visualized in Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gels and by maxicell analysis. ORF464 expressed GDP-mannose pyrophosphorylase and ORF453 encoded a phosphomannomutase, the enzymes for the biosynthesis pathway of GDP-mannose, one of the nucleotide sugar precursors for the formation of the O7 repeating unit. They were designated rfbMEcO7 and rfbKEcO7, respectively. The RfbMEcO7 polypeptide was homologous to the corresponding protein in S. enterica LT2, XanB of Xanthomonas campestris, and AlgA of Pseudomonas aeruginosa, all GDP-mannose pyrophosphorylases. RfbKEcO7 was very similar to CpsG of S. enterica LT2, an enzyme presumably involved in the biosynthesis of the capsular polysaccharide colanic acid, but quite different from the corresponding RfbK protein of S. enterica LT2.

Amino Acid Sequence↗