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PubMed1998

Conserved linkage groups associated with large-scale chromosomal rearrangements between Old World and New World Leishmania genomes.

The genus Leishmania can be taxonomically separated into three main groups: the Old World subgenus L. (Leishmania), the New World subgenus L. (Leishmania) and the New World subgenus L. (Viannia). The haploid genome of Old World Leishmania species has been shown to contain 36 chromosomes defined as physical linkage groups; the latter were found entirely conserved across species. In the present study, we tried to verify whether this conservation of the genome structure extends to the New World species of Leishmania. 300 loci were explored by hybridization on optimized pulsed field gel electrophoresis separations of the chromosomes of polymorphic strains of the six main pathogenic Leishmania species of the New World. When comparing these New World karyotypes with their Old World counterparts, 32 out of 36 linkage groups were found conserved among all species. Four chromosomal rearrangements were found. All species belonging to the L. (Viannia) subgenus were characterized by the presence (i) of a short sequence exchange between chromosomes 26 and 35, and (ii) more importantly, of a fused version of chromosomes 20 and 34 which are separated in all Old World species. 69 additional markers were isolated from a plasmid library specifically constructed from the rearranged chromosomes 20+34 in an attempt to detect mechanisms other than a fusion or breakage: only two markers out of 40 did not belong to the linkage groups 20 and 34. On the other hand, all strains belonging to the New World subgenus L. (Leishmania) were characterized by two different chromosomal rearrangements of the same type (fusion/breakage) as above as compared with Old World species: chromosomes 8+29 and 20+36. Consequently, these two groups of species have 35 and 34 heterologous chromosomes, respectively. Overall, these results show that large-scale chromosomal rearrangements occurred during the evolution of the genus Leishmania, and that the three main groups of pathogenic species are characterized by different chromosome numbers. Nevertheless, translocations seem particularly rare, and the conservation of the major linkage groups should be an essential feature for the compared genetics between species of this parasite.

Animals↗

PubMed2002

Relationship between blaSHV-12 and blaSHV-2a in Korea.

In contrast to the USA and Europe, where SHV-2, SHV-4 and SHV-5 are the prevalent extended-spectrum SHV enzymes, in Korea SHV-2a and SHV-12 are the most frequently identified extended-spectrum SHV enzymes. A 6.6 kb BamHI fragment containing the bla(SHV-12) gene of strain K7746 isolated from one university hospital in Korea was cloned into the pCRScriptCAM vector. Sequencing of the constructed recombinant plasmid pK7746-C1 revealed that the immediate upstream sequence of the bla(SHV-12) gene showed little similarity to the part of the prototype bla(SHV-1) gene due to the insertion of an IS26 element next to the -10 region. Instead, the upstream sequences of bla(SHV-12) retained 100% DNA identity with the part of plasmid pMPA2a from Klebsiella pneumoniae KPZU-3 carrying bla(SHV-2a). The restriction map of the inserted 6.6 kb DNA fragment of plasmid pK7746-C1 was also homologous to that of plasmid pMPA2a, suggesting a common lineage of bla(SHV-12) and bla(SHV-2a). We also studied, using PCR, the upstream non-coding region of several SHV beta-lactamase genes for the presence of IS26 sequence. The flanking IS26 sequence in the immediate upstream region of the bla(SHV) gene was not detected in five standard strains producing SHV-1, SHV-2, SHV-3, SHV-4 or SHV-5. However, IS26 was detected in all 69 clinical strains producing SHV-2a or SHV-12 isolated from three university hospitals in Korea during 1993-1999. The above findings suggest a direct evolution of SHV-12 from SHV-2a, not from SHV-2 to -5, and it is considered to be one of the reasons for the absolute predominance of SHV-2a and SHV-12 in Korea.

Base Sequence↗

PubMed1991

Heterologous in vitro transcription from two archaebacterial promoters.

A cell-free extract of Sulfolobus shibatae is able to specifically initiate transcription in vitro at the promoter of the plasmid-encoded gene for the major gas vesicle protein of Halobacterium halobium and at the promoter for the transcript T4 of the temperate H. halobium phage phi H. The corresponding promoter from the virulent phage mutant phi HL1 yields enhanced transcription in the heterologous system, in agreement with strongly increased in vivo expression.

Archaea↗

PubMed2001

Pathogenicity and resistance islands of staphylococci.

Variable genetic elements including plasmids, transposons and prophages are involved in pathogenesis and antibiotic resistance, and are an important component of the staphylococcal genome. This review covers a set of newly described variable chromosomal elements, pathogenicity and resistance islands, carrying superantigen and resistance genes, especially toxic shock and methicillin resistance, respectively.

Anti-Bacterial Agents↗

PubMed2005

Structural basis for a functional antagonist in the transforming growth factor beta superfamily.

Within the transforming growth factor beta superfamily, the agonist-antagonist relationship between activin and inhibin is unique and critical to integrated reproductive function. Activin acts in the pituitary to stimulate follicle-stimulating hormone, and is antagonized by endocrine acting, gonadally derived inhibin. We have undertaken a mutational analysis of the activin betaA subunit to determine the precise structural aspects that contribute to inhibin antagonism of activin. By substituting specific amino acid residues in the activin betaA subunit with similarly aligned amino acids from the alpha subunit, we have pinpointed the residues required for activin receptor binding and activity, as well as for inhibin antagonism of activin through its receptors. Additionally, we have identified an activin mutant with a higher affinity for the activin type I receptor that provides structural evidence for the evolution of ligand-receptor interactions within the transforming growth factor beta superfamily.

Activin Receptors↗

PubMed1999

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family.

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116. Two copies of IS2112 were found in R. rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the 1-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp. HA1 or in several Rhodococcus strains which do not utilize haloalkanes. IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064, which harbours genes encoding utilization of 1-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA). Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Amino Acid Sequence↗

PubMed1993

Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.

The three classes of enzymes which inactivate aminoglycosides and lead to bacterial resistance are reviewed. DNA hybridization studies have shown that different genes can encode aminoglycoside-modifying enzymes with identical resistance profiles. Comparisons of the amino acid sequences of 49 aminoglycoside-modifying enzymes have revealed new insights into the evolution and relatedness of these proteins. A preliminary assessment of the amino acids which may be important in binding aminoglycosides was obtained from these data and from the results of mutational analysis of several of the genes encoding aminoglycoside-modifying enzymes. Recent studies have demonstrated that aminoglycoside resistance can emerge as a result of alterations in the regulation of normally quiescent cellular genes or as a result of acquiring genes which may have originated from aminoglycoside-producing organisms or from other resistant organisms. Dissemination of these genes is aided by a variety of genetic elements including integrons, transposons, and broad-host-range plasmids. As knowledge of the molecular structure of these enzymes increases, progress can be made in our understanding of how resistance to new aminoglycosides emerges.

Acetyltransferases↗

PubMed2003

Type III intermediate filament proteins interact with four-way junction DNA and facilitate its cleavage by the junction-resolving enzyme T7 endonuclease I.

The isolation from proliferating mouse and human embryo fibroblasts of SDS-stable crosslinkage products of vimentin with DNA fragments containing inverted repeats capable of cruciform formation under superhelical stress and the competitive effect of a synthetic Holliday junction on the binding of cytoplasmic intermediate filament (cIF) proteins to supercoiled DNA prompted a detailed investigation of the proteins' capacity to associate with four-way junction DNA and to influence its processing by junction-resolving endonucleases. Electrophoretic mobility shift analysis of reaction products obtained from vimentin and Holliday junctions under varying ionic conditions revealed efficient complex formation of the filament protein not only with the unstacked, square-planar configuration of the junctions but also with their coaxially stacked X-conformation. Glial fibrillary acidic protein (GFAP) was less efficient and desmin virtually inactive in complex formation. Electron microscopy showed binding of vimentin tetramers or octamers almost exclusively to the branchpoint of the Holliday junctions under physiological ionic conditions. Even at several hundredfold molar excess, sequence-related single- and double-stranded DNAs were unable to chase Holliday junctions from their complexes with vimentin. Vimentin also stimulated bacteriophage T7 endonuclease I in introducing single-strand cuts diametrically across the branchpoint and thus in the resolution of the Holliday junctions. This effect is very likely due to vimentin-induced structural distortion of the branchpoint, as suggested by the results of hydroxyl radical footprinting of Holliday junctions in the absence and the presence of vimentin. Moreover, vimentin, and to a lesser extent GFAP and desmin, interacted with the cruciform structures of inverted repeats inserted into a supercoiled vector plasmid, thereby changing their configuration via branch migration and sensibilizing them to processing by T7 endonuclease I. This refers to both plasmid relaxation caused by unilateral scission and, particularly, linearization via bilateral scission at primary and cIF protein-induced secondary cruciform branchpoints that were identified by T7 endonuclease I footprinting. cIF proteins share these activities with a variety of other architectural proteins interacting with and structurally modulating four-way DNA junctions. In view of the known and hypothetical functions of four-way DNA junctions and associated protein factors in DNA metabolism, cIF proteins as complementary nuclear matrix proteins may play important roles in such nuclear matrix-associated processes as DNA replication, recombination, repair, and transcription, with special emphasis on both the preservation and evolution of the genome.

Bacteriophage T7↗

PubMed1996

Genome size in bacteria.

This manuscript examines genome size in bacteria. The opposing capability of bacteria to alter their genome sizes and order of genes within limits yet remain somewhat constant provides a mechanisms for diversity and evolution in bacterial populations. Bacteria may have evolved by increasing their genome size and changing gene orders with the assistance of restriction endonucleases cleaving foreign DNA and providing a diverse pool of DNA sequences for recombination. These aspects and selected information on physically mapping bacterial genomes will be discussed.

Chromosome Mapping↗

PubMed1999

Class 1 integrons, gene cassettes, mobility, and epidemiology.

Integrons are genetic elements that, although unable to move themselves, contain gene cassettes that can be mobilized to other integrons or to secondary sites in the bacterial genome. The majority of approximately 60 known gene cassettes encode resistance to antibiotics. Recently, a number of gene cassettes encoding extended-spectrum beta-lactamases or carbapenemases have been described. Up to at least five cassettes may be present in an integron, which leads to multiresistance. Frequently, more than one integron is observed within the same bacterial cell. Integrons are widespread in their species distribution. Although integrons are normally reported from Enterobacteriaceae and other gram-negative bacteria, an integron has been described in Corynebacterium glutamicum, a gram-positive species. The gene cassette in this integron showed even higher expression when compared to the expression in Escherichia coli. Integrons have been reported from all continents and are found frequently. The widespread occurrence of integrons is thought to be due to their association with transposon plasmids, conjugative plasmids, or both. Integrons form an important source for the spread of antibiotic resistance, at least in gram-negative bacteria but also potentially in gram-positive bacteria. The aim of this review is to describe the versatility of integrons, especially their mobility and their ability to collect resistance genes.

Animals↗

PubMed1995

Isolation and structure of a new integron that includes a streptomycin resistance gene from the R plasmid of Pseudomonas aeruginosa.

A new integron, located on the R plasmid of Pseudomonas aeruginosa, was isolated in Japan. This integron was made up of two conserved segments (5'-and 3'-conserved segments) and a single streptomycin resistance gene as a gene cassette. The structure of this integron resembles that of integron InC, the existence of which was postulated by Bissonnette and Roy (J. Bacteriol. 174, 1248-1257, 1992).

Biological Evolution↗

PubMed2004

Identification of TFB5, a new component of general transcription and DNA repair factor IIH.

We previously described the use of quantitative proteomics to study macromolecular complexes. Applying the method to analyze a yeast RNA polymerase II preinitiation complex, we identified a new 8-kDa protein, encoded by the uncharacterized open reading frame YDR079c-a, as a potential new component of the preinitiation complex. Here we show that YDR079c-a is a bona fide component of polymerase II preinitiation complexes and investigate its role in transcription. YDR079c-a is recruited to promoters both in vivo and in vitro and is required for efficient transcription in vitro and for normal induction of GAL genes. In addition, YDR079c-a is a core component of general transcription and DNA repair factor IIH and is required for efficient recruitment of TFIIH to a promoter. Yeast lacking YDR079c-a grow slowly, and, like strains carrying mutations in core TFIIH subunits, are sensitive to ultraviolet radiation. YDR079c-a is conserved throughout evolution, and mutations in the human ortholog account for a DNA repair-deficient form of the tricothiodystrophy disorder called TTD-A(2). The identification of a new, evolutionarily conserved, core TFIIH subunit is essential for our understanding of TFIIH function in transcription, DNA repair and human disease.

DNA Polymerase II↗

PubMed1993

Comparative analysis of C3 and botulinal neurotoxin genes and their environment in Clostridium botulinum types C and D.

The C3 exoenzyme gene is located on a bacteriophage in Clostridium botulinum types C and D (M. R. Popoff, D. Hauser, P. Boquet, M. W. Eklund, and D. M. Gill, Infect. Immun. 59:3673-3679, 1991). A derivative CN phage from phage C of C. botulinum Stockholm (C-St) (K. Oguma, H. Iida, and K. Inoue, Jpn. J. Microbiol. 19:167-172, 1975), isolated as neurotoxin negative, also does not produce exoenzyme C3. The botulinal neurotoxin C1 gene is present on the CN phage but contains a stop mutation in the DNA region encoding the N-terminal part of the heavy chain (codon 553). The putative truncated botulinal neurotoxin C1 protein was not recovered in a C. botulinum strain harboring the CN phage. We found that the C3 gene is localized on a 21.5-kbp DNA fragment flanked by the core motif 5'-AAGGAG-3' in DNAs of phage C of C. botulinum 468 (C-468), C-St phage, and phage D of C. botulinum 1873 (D-1873). The 21.5-kbp DNA fragment is deleted in CN phage DNA, and the motif 5'-AAGGAG-3' is present only in one copy at the deletion junction, but the deletion in the CN phage could be nonspecific, since this phage was obtained by nitrosoguanidine treatment. These findings could indicate that the C3 gene is localized on a 21.5-kbp mobile element. C. botulinum type C strain 003-9 produces a C3 exoenzyme (Y. Nemoto, T. Namba, S. Kozaki, and S. Narumiya, J. Biol. Chem. 266:19312-19319, 1991), and Staphylococcus aureus E1 produces a related C3 enzyme which is named epidernmal cell differentiation inhibitor (S. Inoue, M. Sugai, Y. Murooka, S. Y. Paik, Y. M. Hong, H. Oghai, and H. Suginaka, Biochem. Biophys. Res. Comm. 174:459-464, 1991) and which shares 80.6 and 56.6% similarity, respectively with the C3 enzymes from C-468 or C-St and D-1873 phages athe amino acid level. The features of the putative 21.5-kbp transposon were not found in C. botulinum 003-9 and S. aureus E1, as determined by analysis of the C3 and epidermal cell differentiation inhibitor gene-flanking DNA regions. These data suggest a common ancestral origin and divergent evolution of the C3 genes in these three groups of bacterial strains and dissemination of a 21.5-kbp element carrying the C3 gene C-468, C-St, and D-1873 phages.

Amino Acid Sequence↗

PubMed1996

cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors.

cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains). We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene. This pathogenicity island is flanked by direct repeats of 31 bp. In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605). In a minority of H. pylori strains, cagI and cagII are separated by an intervening chromosomal sequence. Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3' end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens. Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines. Thus, we believe the cag region may encode a novel H. pylori secretion system for the export of virulence determinants.

Antigens, Bacterial↗

PubMed2005

[Heterogenous expression of nitrile hydratase gene of Nocardia sp. in recombinant Pichia pastoris].

Heterogenous expression of active nitrile hydratase (NHase) was focused for its great potential in genetically evolution of the operational stability of NHase. Two recombinant Escherichia coli strains, E. coli JM105 (pUC18-NHBAX) and E. coli BL21 (DE3) (pET32a-NHBAX), were first constructed and used for heterogenous expression of a NHase gene cloned from Nocardia YS-2002. It was found that the alpha subunit of NHase can not be effectively expressed in both recombinant E. coli, which results in as low as 0.04U/mg (dry cell weight) activity of NHase in E. coli BL21 (DE3) (pET32a-NHBAX), and no activity in JM105 (pUC18-NHBAX) at all. Therefore, the recombinant and active expression of NHase in Pichia pastoris was especially interested. A new plasmid, pPIC3.5k-NHBAX, was constructed by insertion of the NHase gene, and then successfully transformed into the cell of Pichia pastoris GS115 by electroporation. A novel superior strain, P. pastoris NH4, was selected from 6 target-clones and used for optimization of cell culture and NHase expression. The final activity of NHase expressed in P. pastoris NH4 under optimal conditions is 0.52U/mg (dry cell weight), which is 13-fold higher than that in recombinant E. coli. However, the activity of NHase expressed in recombinant P. pastoris NH4 can not be stably maintained longer than 6 h.

Enzyme Induction↗

PubMed1999

Specificity determinants of proteolytic processing of Aspergillus PacC transcription factor are remote from the processing site, and processing occurs in yeast if pH signalling is bypassed.

The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.

Amino Acid Sequence↗

PubMed1990

Expression in Escherichia coli of the psbO gene encoding the 33 kd protein of the oxygen-evolving complex from spinach.

The cDNA for the 33 kd protein from the oxygen-evolving complex of spinach together with the coding region for the hydrophobic C-terminal part of the transit sequence was cloned into the expression plasmid pDS12/33Ex. The 33 kd protein precursor was expressed in Escherichia coli, secreted into the periplasm and correctly processed to the mature 33 kd protein. Thus the hydrophobic domain of the transit sequence, preceded by a methionine and two lysine residues, can function as a bacterial signal peptide. The periplasmic proteins were released from the cells by osmotic shock and the expressed protein was purified by anion exchange chromatography. The protein was identified by SDS-PAGE and Western blotting. N-terminal sequence analysis showed that the cleavage of the signal peptide occurred at the correct position. The expressed protein could be rebound to CaCl2-washed PSII particles and oxygen evolution was restored in equal amounts by the 33 kd protein from both E. coli and spinach.

Amino Acid Sequence↗

PubMed1998

Genotypes of Helicobacter pylori in Lithuanian families.

BACKGROUND: Infection by Helicobacter pylori is very common in Eastern Europe, but the genotypes of predominant strains and prevalence of single vs. multiple infection in this geographic region have not been much studied. MATERIALS AND METHODS: H. pylori was cultured from 13 Lithuanians belonging to six families, and characterized by arbitrarily primed PCR (RAPD) DNA fingerprinting, and by hybridization and PCR tests for polymorphic virulence-associated and neutral genetic markers. RESULTS: Eleven distinct strains were identified: seven carried the cag pathogenicity island (PAI) and the s1 (generally toxigenic) allele of the vacuolating cytotoxin gene (vacA); the other four were cag- and carried the vacA s2 (nontoxigenic) allele; five of the seven vacA s1 strains carried an m1 middle region allele of vacA, whereas all other strains carried m2 alleles, which are generally less toxigenic; four strains carried the virulence-associated iceA1 gene, and the other seven carried the completely unrelated iceA2 gene at the same locus. Insertion sequences IS605 and IS606 and a plasmid replication gene (repA) were also found in some strains. RAPD fingerprinting identified a mixed infection in just one of the 13 persons. In two families, two of the members harbored the same strain, whereas in the other four families each member tested carried a different strain. Resistance to metronidazole (Mtz) was found in two persons; each of them also carried MtzS strains that were indistinguishable from the coresident MtzR strain by RAPD fingerprinting, and that were thus closely related in overall genotype. CONCLUSION: The distribution of genotypes of Lithuanian H. pylori strains resembles that seen in Western Europe. This finding has important implications for understanding modes of H. pylori transmission and evolution.

Adolescent↗