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PubMed1989

Molecular evolution of enzyme structure: construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase.

Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. In Escherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster and E. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of the E. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the "polar domain") of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the "equatorial domain") was derived from a cloned pyrBI operon of E. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformed E. coli pyrB- cells. The functionality of this E. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.

Amino Acid Sequence↗

PubMed2003

Analysis of genomic diversity and evolution of the low-level antiseptic resistance gene smr in Staphylococcus aureus.

A multidrug efflux pump specified by the smr gene mediates low-level antiseptic resistance in staphylococci. We analyzed the genomic diversity of smr and its gene cassette, a structural unit containing smr and terminal direct repeats (DRs), in 22 clinical strains of Staphylococcus aureus isolated over 9 years in a Japanese hospital. Although open reading frames (ORFs) of all the smr genes examined were identical to those reported previously (e.g., qacD in pSK41), smr gene cassettes were classified into three groups (types 1, 2, and 3). The type 1 cassette had an identical genetic organization to that found in the plasmid pSK41, a putative prototype of the smr gene cassette, which contains DRs flanking smr. In the type 2 cassette, the rep gene and a putative replication nick site were found upstream of smr, between a SSOA (single-strand origin) sequence and DR1c, which are components of the type 1 cassette DR. In the type 3 cassette detected in a single strain, IS431 was located between the 3' end of smr and DR. It was suggested by genomic comparison that type 2 and type 3 cassettes might have been derived from the type 1 cassette via insertion of foreign DNA sequence, and that the type 2 cassette might be a precursor form of some previously reported smr cassettes, such as those in pSK108 and pNVH99. Although MICs of antiseptics and ethidium bromide were generally the same among strains having type 1, 2, or 3 smr gene cassette, the type 1 cassette was detected most frequently. Moreover, the copy number of the smr gene in the type 2 cassette was found to be much higher than that in the type 1 or type 3 cassette.

Anti-Bacterial Agents↗

PubMed1987

Rearrangement of duplicated DNA in specialized cells of Neurospora.

Introduction of DNA into Neurospora crassa can lead to sequence instability in the sexual phase of the life cycle. Sequence instability was investigated by using a set of strains transformed with single copies of a plasmid including host sequences, Neurospora sequences deleted from the host genome, and foreign sequences. The sequences already represented in the host were rearranged at high frequency in a cross. In general, both elements of the duplication, that from the plasmid and that from the host, became rearranged, whether or not they were linked. Unique sequences were left unaltered. Cytosine residues in the rearranged sequences typically became methylated de novo. Results from tetrad analyses indicated that the rearrangements occur before meiosis, during a stage between fertilization and karyogamy. We suggest that this previously unrecognized genetic process, RIP (rearrangement induced premeiotically), may contribute diversity for evolution and also maintain the gross organization of the genome.

DNA Replication↗

PubMed2004

Evolution of vitamin B2 biosynthesis: structural and functional similarity between pyrimidine deaminases of eubacterial and plant origin.

The Arabidopsis thaliana open reading frame At4g20960 predicts a protein whose N-terminal part is similar to the eubacterial 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate deaminase domain. A synthetic open reading frame specifying a pseudomature form of the plant enzyme directed the synthesis of a recombinant protein which was purified to apparent homogeneity and was shown by NMR spectroscopy to convert 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate into 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate at a rate of 0.9 micromol mg(-1) min(-1). The substrate and product of the enzyme are both subject to spontaneous anomerization of the ribosyl side chain as shown by (13)C NMR spectroscopy. The protein contains 1 eq of Zn(2+)/subunit. The deaminase activity could be assigned to the N-terminal section of the plant protein. The deaminase domains of plants and eubacteria share a high degree of similarity, in contrast to deaminases from fungi. These data show that the riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a different pathway.

Amino Acid Sequence↗

PubMed1993

Iterative chromosome mutation and selection as a mechanism of complete transformation of human diploid fibroblasts by SV40 T antigen.

The acquisition of an extended lifespan and of neoplastic properties, including anchorage independence, ability to grow in low serum-containing media, morphological transformation, immortalization and tumorigenicity in nude mice were studied in 31 human fibroblast lineages transfected with plasmids containing the SV40 early genes. Plasmids were used that contained sequences for large T alone, or large T plus small t or large T plus small t plus the SV40 origin. Cells expressing large T antigen gradually acquired the ability to form colonies in low serum or to form anchorage-independent colonies. Large T antigen was sufficient to cause complete transformation to tumorigenicity if multi-step lineage evolution was obtained by prolonged serial passage and if in vivo progression was assisted by means of a gelatin sponge implantation technique. Cells derived from progressive tumors initiated in sponges showed enhanced tumorigenicity as measured by ability to obtain tumors without using sponges and with reduced latent period, higher incidence and with fewer cells inoculated. Multiple lineages of human fibroblasts have been converted to tumorigenicity without additional treatments such as transfection with activated oncogenes or exposure to carcinogens. These data, taken in conjunction with earlier studies showing that T antigen causes chromosome mutation preceding and accompanying the accumulation of the neoplastic phenotype, suggests that the T protein drives the transformation process by acting as a mutagen and cells with growth advantages were selected for in vitro and in vivo. With the possible exception of morphological transformation, the presence or absence of genes for small t and the SV40 origin were not critical for the process.

Antigens, Polyomavirus Transforming↗

PubMed2005

Genome dynamics and diversity of Shigella species, the etiologic agents of bacillary dysentery.

The Shigella bacteria cause bacillary dysentery, which remains a significant threat to public health. The genus status and species classification appear no longer valid, as compelling evidence indicates that Shigella, as well as enteroinvasive Escherichia coli, are derived from multiple origins of E.coli and form a single pathovar. Nevertheless, Shigella dysenteriae serotype 1 causes deadly epidemics but Shigella boydii is restricted to the Indian subcontinent, while Shigella flexneri and Shigella sonnei are prevalent in developing and developed countries respectively. To begin to explain these distinctive epidemiological and pathological features at the genome level, we have carried out comparative genomics on four representative strains. Each of the Shigella genomes includes a virulence plasmid that encodes conserved primary virulence determinants. The Shigella chromosomes share most of their genes with that of E.coli K12 strain MG1655, but each has over 200 pseudogenes, 300 approximately 700 copies of insertion sequence (IS) elements, and numerous deletions, insertions, translocations and inversions. There is extensive diversity of putative virulence genes, mostly acquired via bacteriophage-mediated lateral gene transfer. Hence, via convergent evolution involving gain and loss of functions, through bacteriophage-mediated gene acquisition, IS-mediated DNA rearrangements and formation of pseudogenes, the Shigella spp. became highly specific human pathogens with variable epidemiological and pathological features.

DNA Transposable Elements↗

PubMed1989

[Drug-resistance and its transferability of Shigella strains isolated in 1986 in Japan].

Two hundred and fifty Shigella strains isolated in Japan were examined for their drug-resistances and their conjugal transferability. Drugs used and the isolation frequency of resistant strains were: sulfanilamide (SA) 96%, streptomycin (SM) 85%, tetracycline (TC) 82%, fosfomycin (FOM) 76%, chloramphenicol (CP) 51%, trimethoprim (TMP) 46%, ampicillin (ABPC) 45%, nalidixic acid (NA) 6%, kanamycin (KM) 5%, cephaloridine (CER) 4%, gentamicin (GM) 0%, amikacin (AMK) 0%, colistin (CL) 0%, norfloxacin (NFLX) 0%. All of the KM-resistances and none of the FOM-resistances were transferable. Each 20 to 30% of CP, TC, SM, SA or ABPC-resistant isolates transferred each drug-resistance. Two thirds of the isolates were brought to Japan by travellers mostly from South-East Asia. The isolation frequency of resistant strains was not different between domestic and foreign strains except that NA-resistance was not found in foreign strains. The frequency of isolates carrying transferable R plasmids were higher in domestic strains than foreign strains. Most of the isolated had multiple drug-resistance. Major resistance types were: (CP, TC, SM, SA, ABPC) 22%; (TC, SM, SA, TMP) 20%; (CP, TC, SM, SA, ABPC, TMP) 16%; (TC, SM, SA) 8%. Strains showing different resistance types were different among each other in their serum type, conjugal transferability, and derived country. Stepwise evolution of multiple resistance in one country was not demonstrated. The resistance types of Shigella isolates in Japan have been influenced by yearly change of imported strains.

Anti-Bacterial Agents↗

PubMed2000

Transfer RNA identity change in anticodon variants of E. coli tRNA(Phe) in vivo.

The anticodon sequence is a major recognition element for most aminoacyl-tRNA synthetases. We investigated the in vivo effects of changing the anticodon on the aminoacylation specificity in the example of E. coli tRNA(Phe). Constructing different anticodon mutants of E. coli tRNA(Phe) by site-directed mutagenesis, we isolated 22 anticodon mutant tRNA(Phe); the anticodons corresponded to 16 amino acids and an opal stop codon. To examine whether the mutant tRNAs had changed their amino acid acceptor specificity in vivo, we tested the viability of E. coli strains containing these tRNA(Phe) genes in a medium which permitted tRNA induction. Fourteen mutant tRNA genes did not affect host viability. However, eight mutant tRNA genes were toxic to the host and prevented growth, presumably because the anticodon mutants led to translational errors. Many mutant tRNAs which did not affect host viability were not aminoacylated in vivo. Three mutant tRNAs containing anticodon sequences corresponding to lysine (UUU), methionine (CAU) and threonine (UGU) were charged with the amino acid corresponding to their anticodon, but not with phenylalanine. These three tRNAs and tRNA(Phe) are located in the same cluster in a sequence similarity dendrogram of total E. coli tRNAs. The results support the idea that such tRNAs arising from in vivo evolution are derived by anticodon change from the same ancestor tRNA.

Anticodon↗

PubMed1991

Cloning and characterization of a chromosomal DNA region required for growth on 2,4,5-T by Pseudomonas cepacia AC1100.

A series of spontaneous 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) nonmetabolizing mutants of Pseudomonas cepacia AC1100 were characterized to be defective in either 2,4,5-T uptake or conversion of this compound to 2,4,5-trichlorophenol (2,4,5-TCP). Two of these mutants, RHC22 and RHC23, were complemented for growth on 2,4,5-T using an AC1100 genomic library constructed in the cosmid vector pCP13. Recombinant cosmids isolated from the complemented mutants contained a 27.5-kb insert which frequently underwent various-sized deletions in Escherichia coli. Hybridization studies showed this DNA to be of chromosomal origin and totally deleted in RHC22, RHC23 and other similar mutants. Complementation analyses of RHC22 with a series of subcloned fragments and spontaneously deleted derivatives of the recombinant cosmid pRHC21 showed the 2,4,5-T (tft) genes to occur within an 8.9-kb region. Pseudomonas aeruginosa cells transformed with this DNA acquired the ability to convert 2,4,5-T to 2,4,5-TCP. The genetic determinant for this function was further localized within a 3.7-kb region. This DNA, in the absence of other sequences from the 8.9-kb tft gene region allowed RHC22 cells to metabolize 2,4,5-T, but at low rates which were insufficient to support growth. Copies of the insertions sequence element IS931 were identified either adjacent to or within this tft gene region in the genomes of two independent wild-type AC1100 isolates. Preliminary evidence suggests that these sequences either facilitate or are required for growth on 2,4,5-T and hence may be implicated in the genetic evolution of the 2,4,5-T metabolic pathway.

2,4,5-Trichlorophenoxyacetic Acid↗

PubMed2001

Identification of a mutation causing increased expression of the tas gene in Escherichia coli FX-11.

Studies of N-ethyl-N-nitrosourea (ENU)-induced mutagenesis with a tyrosine auxotroph of Escherichia coli revealed a new type of revertant. This mutant strain was interesting because: (i) it was not a true revertant of the nonsense (ochre) defect nor a tRNA suppressor mutation; and (ii) it was induced by ENU to greater extent in a UmuC-defective host. Genetic mapping located the probable mutation to a region of the E. coli chromosome containing a newly described gene called tas. To investigate this mutation, the upstream region of the tas gene from both wild-type and mutant cells was cloned into a promoterless lacZ expression vector and recombined onto a lambda bacteriophage. Recombinant bacteriophage were inserted into the bacterial chromosome and beta-galactosidase (betaGal) assays were performed. These assays revealed an almost three-fold greater expression of betaGal from the mutant DNA than from the wild-type DNA. Sequence analysis of the region directly upstream of the tas gene revealed a G:C to A:T transition at base number 2263 (numbering based on GenBank Accession #AE000367), located within a potential promoter site. Further sequencing indicated no other mutations within the 1454bp region analyzed; however, there were several nucleotide differences seen in our B/r strain of E. coli, when compared with the published E. coli K-12 sequence. A total of 10 base differences were discovered; one in mutH, six within a potential open reading frame (ORF-o237) and three in non-coding regions. Yet, none of the changes altered the predicted amino acid sequences. These results provide evidence of a mechanism for increased expression of the novel gene tas and support the neutral drift hypothesis for the evolution of DNA sequences.

Amino Acid Sequence↗

PubMed2001

Box C/D RNA guides for the ribose methylation of archaeal tRNAs. The tRNATrp intron guides the formation of two ribose-methylated nucleosides in the mature tRNATrp.

Following a search of the Pyrococcus genomes for homologs of eukaryotic methylation guide small nucleolar RNAs, we have experimentally identified in Pyrococcus abyssi four novel box C/D small RNAs predicted to direct 2'-O-ribose methylations onto the first position of the anticodon in tRNALeu(CAA), tRNALeu(UAA), elongator tRNAMet and tRNATrp, respectively. Remarkably, one of them corresponds to the intron of its presumptive target, pre-tRNATrp. This intron is predicted to direct in cis two distinct ribose methylations within the unspliced tRNA precursor, not only onto the first position of the anticodon in the 5' exon but also onto position 39 (universal tRNA numbering) in the 3' exon. The two intramolecular RNA duplexes expected to direct methylation, which both span an exon-intron junction in pre-tRNATrp, are phylogenetically conserved in euryarchaeotes. We have experimentally confirmed the predicted guide function of the box C/D intron in halophile Haloferax volcanii by mutagenesis analysis, using an in vitro splicing/RNA modification assay in which the two cognate ribose methylations of pre-tRNATrp are faithfully reproduced. Euryarchaeal pre-tRNATrp should provide a unique system to further investigate the molecular mechanisms of RNA-guided ribose methylation and gain new insights into the origin and evolution of the complex family of archaeal and eukaryotic box C/D small RNAs.

Base Sequence↗

PubMed1990

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria.

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.

Acetyl-CoA C-Acyltransferase↗

PubMed2005

Dissemination of transferable CTX-M-type extended-spectrum beta-lactamase-producing Escherichia coli in Korea.

AIMS: Among 365 Escherichia coli isolated in 2003, 31 cefotaxime-resistant isolates were obtained from clinical specimens taken from adults hospitalized in Busan, Korea. Six extended-spectrum beta-lactamase (ESBL)-producing isolates were investigated further to determine the mechanism of resistance. METHODS AND RESULTS: These isolates were analysed by antibiotic susceptibility testing, pI determination, plasmid profiles, transconjugation test, PCR-restriction fragment length polymorphism (RFLP), enterobacterial repetitive consensus (ERIC)-PCR and DNA sequencing. All six of these isolates were found to contain the CTX-M-type ESBL genes. Five clinical isolates and their transconjugants produced CTX-M-3. One clinical isolate (K17391) and its transconjugant (trcK17391) produced CTX-M-15. Five clinical isolates also produced another TEM-1. One clinical isolate (K12776) also contained another TEM-52. CTX-M-3 ESBL gene was responsible for the resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam. CTX-M-15 or TEM-52 was especially responsible for the resistance to ceftazidime. CONCLUSIONS: These results appear to represent the in vivo evolution of CTX-M-type beta-lactamase genes (bla(CTX-M-3) --> bla(CTX-M-15)) under the selective pressure of antimicrobial therapy (especially ceftazidime). PCR-RFLP is a reliable method to discriminate CTX-M-15 gene from CTX-M-3 gene. ERIC-PCR analysis revealed that dissemination of CTX-M-3 was not due to a clonal outbreak of a resistant strain but to the intra-species spread of resistance to piperacillin, cephalothin, cefotaxime, cefepime and aztreonam in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the occurrence of CTX-M-1 cluster ESBLs in Korea. A more comprehensive survey of these ESBL types from Korea is urgently needed because of the in vivo evolution of CTX-M-15 from CTX-M-3. The emergence of these CTX-M-type ESBLs suggests that diagnostic laboratories should screen for ESBLs with ceftazidime as well as cefotaxime; they should still perform clavulanate synergy tests on resistant isolates.

Adult↗

PubMed1993

Responsible use of antimicrobials in veterinary medicine: perspectives in France.

In France a survey of the use of antimicrobials in veterinary medicine was set up in connection with the Committee for the Marketing Authorization of Veterinary Medicinal Products. This survey has emphasized the importance of antimicrobials in veterinary medicine and the reality of antibiotic resistance in both man and animals. Certain epidemiological data tend to demonstrate that the use of antibiotics in animals increases the risk of antibiotic resistance in man, through selection in animals of resistant pathogenic bacteria or of a non-pathogenic resistant bacteria carrying resistance plasmids. Four proposals are put forward for the control of these different risks: reserve certain antibiotics (life-saving antibiotics) for human medicine, conform to a code of good practices for antibiotic use in animals, strengthen and connect up existing networks for the epidemiosurveillance of antibiotic resistance, provide for studies on the ecological impact of antibiotics in the registration dossiers for veterinary medicinal products. These evolutive measures, based on scientific progress in microbiology, epidemiology and clinical studies should respond satisfactorily to the expectations of all concerned.

Animals↗

PubMed2006

Exopolysaccharide expression in Lactococcus lactis subsp. cremoris Ropy352: evidence for novel gene organization.

Lactococcus lactis subsp. cremoris Ropy352 produces two distinct heteropolysaccharides, phenotypically described as ropy and mucoid, when cultured in nonfat milk. One exopolysaccharide precipitated with 50% ethanol as a series of elongated threads and was composed of glucose and galactose in a molar ratio of 3:2. The second exopolysaccharide precipitated with 75% ethanol as a fine flocculant and consisted of galactose, glucose, and mannose with a molar ratio of 67:21:12. A mutant strain, L. lactis subsp. cremoris EK240, lacking the ropy phenotype did not produce the exopolysaccharide that precipitated with 50% ethanol; however, it produced the exopolysaccharide that precipitated with 75% ethanol, indicating that the former exopolysaccharide is essential for the ropy phenotype. Cultures of L. lactis subsp. cremoris Ropy352 in 10% nonfat milk reached a viscosity of 25 Pa-s after 24 h, while those of the nonropy L. lactis subsp. cremoris EK240 mutant did not change. A mutation abolishing ropy exopolysaccharide expression mapped to a region on a plasmid containing two open reading frames, epsM and epsN, encoding novel glycosyltransferases bordered by ISS1 elements oriented in the same direction. Sequencing of this plasmid revealed two other regions involved in exopolysaccharide expression, an operon located between partial IS981 and IS982 elements, and an independent gene, epsU. Two and possibly three of these regions are involved in L. lactis subsp. cremoris Ropy352 exopolysaccharide expression and are arranged in a novel fashion different from that of typical lactococcal exopolysaccharide loci, and this provides genetic evidence for exopolysaccharide gene reorganization and evolution in Lactococcus.

Animals↗

PubMed2002

The superoxide dismutase gene sodM is unique to Staphylococcus aureus: absence of sodM in coagulase-negative staphylococci.

Superoxide dismutase (SOD) profiles of clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) were determined by using whole-cell lysates and activity gels. All S. aureus clinical isolates exhibited three closely migrating bands of activity as previously determined for laboratory strains of S. aureus: SodM, SodA, and a hybrid composed of SodM and SodA (M. W. Valderas and M. E. Hart, J. Bacteriol. 183:3399-3407, 2001). In contrast, the CoNS produced only one SOD activity, which migrated similarly to SodA of S. aureus. Southern analysis of eight CoNS species identified only a single sod gene in each case. A full-length sod gene was cloned from Staphylococcus epidermidis and determined to be more similar to sodA than to sodM of S. aureus. Therefore, this gene was designated sodA. The deduced amino acid sequence of the S. epidermidis sodA was 92 and 76% identical to that of the SodA and SodM proteins of S. aureus, respectively. The S. epidermidis sodA gene expressed from a plasmid complemented a sodA mutation in S. aureus, and the protein formed a hybrid with SodM of S. aureus. Both hybrid SOD forms as well as the SodM and SodA proteins of S. aureus and the S. epidermidis SodA protein exist as dimers. These data indicate that sodM is found only in S. aureus and not in the CoNS, suggesting an important divergence in the evolution of this genus and a unique role for SodM in S. aureus.

Amino Acid Sequence↗

PubMed2006

The clc element of Pseudomonas sp. strain B13, a genomic island with various catabolic properties.

Pseudomonas sp. strain B13 is a bacterium known to degrade chloroaromatic compounds. The properties to use 3- and 4-chlorocatechol are determined by a self-transferable DNA element, the clc element, which normally resides at two locations in the cell's chromosome. Here we report the complete nucleotide sequence of the clc element, demonstrating the unique catabolic properties while showing its relatedness to genomic islands and integrative and conjugative elements rather than to other known catabolic plasmids. As far as catabolic functions, the clc element harbored, in addition to the genes for chlorocatechol degradation, a complete functional operon for 2-aminophenol degradation and genes for a putative aromatic compound transport protein and for a multicomponent aromatic ring dioxygenase similar to anthranilate hydroxylase. The genes for catabolic functions were inducible under various conditions, suggesting a network of catabolic pathway induction. For about half of the open reading frames (ORFs) on the clc element, no clear functional prediction could be given, although some indications were found for functions that were similar to plasmid conjugation. The region in which these ORFs were situated displayed a high overall conservation of nucleotide sequence and gene order to genomic regions in other recently completed bacterial genomes or to other genomic islands. Most notably, except for two discrete regions, the clc element was almost 100% identical over the whole length to a chromosomal region in Burkholderia xenovorans LB400. This indicates the dynamic evolution of this type of element and the continued transition between elements with a more pathogenic character and those with catabolic properties.

Aminophenols↗

PubMed2004

[Study on the reconstitution in vitro and photochemical activities of phytochrome from the Synechocystis sp. PCC6803].

Genomic DNA sequence analysis of phytochrome like photoreceptors in a number of bacteria revealed several open reading frames (ORFs) encoding proteins with amino acid sequences homologous to plant phytochromes. The phytochrome like photoreceptors, collectively called bacteriophytochromes, contain an N-terminal domain homologous to the chromophore-binding domain (CBD) of higher plants and a C-terminal domain of histidine kinase domain( HKD). Due to their simple structure, bacteriophytochromes broaden the view of phytochrome evolution and provide us with a simple model to investigate phytochrome-mediated light signal in higher plants. In this report, the bacteriophytochromes from Synechocystis sp. PCC6803 were investigated. The gene cph1 and its fragment cph1 (C-435) were isolated from the Synechocystis sp. PCC6803 genomic DNA by polymerase chain reaction(PCR) using specific primers. Then, the genes were cloned with the vector pBluescript, yielding plasmids pBlu-cphl and pBlu-cph1 ( C-435), before they are subcloned with the vector pET30, using the EcoRV and Xho I restriction sites. pBlu-cph1, pBlu-cph1 (N-435) were cleaved with Sma I and Xho I, and the released genes were ligated to the pET30a fragment. The E. coli [strain BL21 (DE3)] cells containing recombinant pET30a were grown in medium RB at 20 degrees C, and harvested 6 h later after induction with isopropyl thio-beta-D-galactoside (IPTG). Then, reconstitution systems were employed to study the characteristics of the genes. In the reconstitution system, autoassembly of aprotein of phytochrome with PCB was investigated. The chromophore addition was an autocatalytic process. Reconstitution products were red/infrared (R/FR) photochromic, which was similar to that of the phytoehrome in higher plants. How ever, the spectral change ratios (deltaAmax/deltaAmin) of the two fragments differed from each other. It was also shown that PCB was covalently bound to apo-protein via Zn2+ fluoresc ence SDS-PAGE. After irradiation by light of 700 nm, the maximum absorption spectrum o f holo-Cphl was 650nm. The absorption of it after denaturatior in the dark with ur ea in the presence of hydrochloric acid (pH = 2) was 660nm, which was similar with th at of cis-PCB. In addition, after irradiation by light of 650nm, the maximum absorption spectrum of holo-Cph1 was 700nm. The absorption of it after denaturation in the dark with urea in the presence of hydrochloric acid (pH = 2) was 600nm, which was similar with that of trans-PCB. The result showed that the photochromism of phytochrome resulted from the isomerizaation of chromophore (PCB in this report). The reconstitution of Cph1 (C-435) under the same condition supported the conclusion. Fluorescence emission spectrum of the products suggested that bacteriophytochrom e structure with cis-PCB was more stable than that with trans-PCB. The new reconstitution system in this report sets a base for the application of phytochrome as photochromic biomaterials in biosensors. In addition, phytochrome shows great potential in food, cosmetic and biological engineering, etc.

Bacterial Proteins↗