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PubMed2005

Identification of functional domains within the bICP0 protein encoded by bovine herpesvirus 1.

It is believed that the bICP0 protein encoded by bovine herpesvirus 1 (BoHV-1) stimulates productive infection by activating viral gene expression. Like the other ICP0-like proteins encoded by alphaherpesvirinae subfamily members, bICP0 contains a zinc RING finger near its amino terminus. The zinc RING finger of bICP0 activates viral transcription, stimulates productive infection, and is toxic to certain cell types. Apart from the zinc RING finger, bICP0 possesses little similarity to the herpes simplex virus type 1 ICP0 protein making it difficult to predict what regions of bICP0 are important. To begin to identify bICP0 functional domains that are not part of the zinc RING finger, a panel of transposon insertion mutants that span bICP0 was developed. A large domain spanning aa 78-256, and a separate domain that is at or near aa 457 was necessary for efficient transactivation of a simple promoter. Transposon insertion at aa 91 impaired bICP0 protein stability in transfected cells. Insertion of transposons into the acidic domain of bICP0 had little or no effect on transactivation of a simple promoter or protein expression suggesting this region does not play a major role in activating gene expression. Sequences near the C terminus (aa 607-676) contain a functional nuclear localization signal. Collectively, these studies indicated that bICP0 contains several important functional domains: (i) the zinc RING finger, (ii) two separate domains that activate transcription, and (iii) a C-terminal nuclear localization signal that is also necessary for efficient transactivation.

Amino Acid Sequence↗
PubMed1996

Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA.

Pyrazinamide (PZA) is one of the three most important drugs for treatment of Mycobacterium tuberculosis infections. The antibacterial activity of PZA requires a bacterial enzyme, pyrazinamidase (PZAase), which hydrolyzes PZA to form pyrazinoic acid and ammonia. Most PZA-resistant clinical M. tuberculosis isolates lack PZAase activity. With the goal of eventually identifying and characterizing the M.tuberculosis PZAase gene, we began with the more tractable organism, Escherichia coli, which also has PZAase activity. We screened a transposon-generated E. coli insertion mutant library, using a qualitative PZAase assay. Two PZAase-negative mutants out of 4,000 colonies screened were identified. In each mutant, the transposon interrupted the same 639-bp open reading frame (ORF), ORF1. The expression of ORF1 on a multicopy plasmid complemented a PZAase-negative mutant, leading to PZAase activity levels approximately 10-fold greater than those of the wild type. PZA has a structure similar to that of nicotinamide, a pyridine nucleotide cycle intermediate, so we tested our strains for nicotinamidase activity (EC 3.5.1.19) (genetic locus pncA). The construct with multiple plasmid copies of ORF1 had an approximately 10-fold increase in levels of nicotinamidase activity. This overexpressing strain could utilize nicotinamide as a sole nitrogen source, through wild-type E. coli cannot. We conclude that a single E. coli enzyme accounts for both PZAase and nicotinamidase activities and that ORF1 is the E.coli PZAase and nicotinamidase gene, pncA.

Amidohydrolases↗
PubMed1990

Identification of an iron-regulated virulence determinant in Vibrio cholerae, using TnphoA mutagenesis.

Several virulence determinants of bacteria are regulated by the concentration of iron in the medium, with increased expression occurring under low-iron conditions. Iron-regulated virulence factors have not been previously described in Vibrio cholerae. We used the transposon vector Tn5 IS50L::phoA (TnphoA) to obtain insertion mutations in iron-regulated genes of V. cholerae 0395. One strain, carrying an insertion mutation in iron-regulated gene irgA, had reduced virulence in an animal model and had lost the 77-kilodalton major iron-regulated outer membrane protein. The 50% lethal dose of this mutant strain (MBG40) in suckling mice was 3 x 10(5) bacteria compared with 4 x 10(3) bacteria for 0395 wild type. In an in vivo competition assay, the wild-type strain out competed the mutant almost 10-fold, suggesting a possible colonization defect in MBG40. The in vitro competitive index and in vitro growth curves in low- and high-iron media showed that the mutant strain had no discernable growth defect. These data suggest that the 77-kilodalton iron-regulated outer membrane protein may play a role in the pathogenesis of V. cholerae infection. It is not yet clear whether the reduction of virulence in MBG40, as assessed by 50% lethal dose analysis, is explained by a colonization defect or whether it relates to another defect associated with loss of the IrgA protein.

Alkaline Phosphatase↗
PubMed2000

Identification of genes associated with copper tolerance in an adhesion-defective mutant of Aeromonas veronii biovar sobria.

TnphoA mutagenesis was used to identify adhesins of Aeromonas veronii biovar sobria 3767, a strain isolated from a diarrhoeal stool specimen. Six mutants, from a library of 154, exhibited significantly reduced levels of adhesion to HEp-2 cells. Primers to the terminal regions of TnphoA were used for inverse PCR and the product from one mutant was cloned into pBluescript and partial sequence data obtained. Scanning GenBank and EMBL data bases revealed DNA sequence similarity to the copA gene of Pseudomonas syringae pv. tomato which confers resistance to copper and other heavy metals. The transposon was located within the copA gene and the mutant exhibited a reduced tolerance to copper. Primer walking, using the inverse PCR product as a template, revealed three open reading frames (ORFs) copA, B and C in A. veronii biovar sobria 3767. The predicted amino acid sequences of ORFs A and B had significant homology (55 and 34% respectively) to the copA and B proteins of P. syringae. No amino acid or DNA sequence homology existed between ORF C of strain 3767 and any other gene in the data bases scanned. Further analysis of the nucleotide sequence failed to reveal the presence of typical copper regulatory genes within the vicinity of the Aeromonas sequence. The association between copper tolerance and adhesion in A. veronii biovar sobria requires further study.

Adhesins, Bacterial↗
PubMed2006

Identification of Burkholderia pseudomallei genes required for the intracellular life cycle and in vivo virulence.

The bacterial pathogen Burkholderia pseudomallei invades host cells, escapes from endocytic vesicles, multiplies intracellularly, and induces the formation of actin tails and membrane protrusions, leading to direct cell-to-cell spreading. This study was aimed at the identification of B. pseudomallei genes responsible for the different steps of this intracellular life cycle. B. pseudomallei transposon mutants were screened for a reduced ability to form plaques on PtK2 cell monolayers as a result of reduced intercellular spreading. Nine plaque assay mutants with insertions in different open reading frames were selected for further studies. One mutant defective in a hypothetical protein encoded within the Bsa type III secretion system gene cluster was found to be unable to escape from endocytic vesicles after invasion but still multiplied within the vacuoles. Another mutant with a defect in a putative exported protein reached the cytoplasm but exhibited impaired actin tail formation in addition to a severe intracellular growth defect. In four mutants, the transposon had inserted into genes involved in either purine, histidine, or p-aminobenzoate biosynthesis, suggesting that these pathways are essential for intracellular growth. Three mutants with reduced plaque formation were shown to have gene defects in a putative cytidyltransferase, a putative lipoate-protein ligase B, and a hypothetical protein. All nine mutants proved to be significantly attenuated in a murine model of infection, with some mutants being essentially avirulent. In conclusion, we have identified a number of novel major B. pseudomallei virulence genes which are essential for the intracellular life cycle of this pathogen.

4-Aminobenzoic Acid↗
PubMed1989

Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes.

Biochemical analysis of the enzymatic activity catalyzing the conversion of chorismate to isochorismate in the enterobactin biosynthetic pathway attributed the reaction to the isochorismate synthetase enzyme, designated EntC. However, the lack of mutations defining this activity has hampered the precise identification of the entC structural gene. In this study, we engineered a stable insertion mutation into the chromosomal region between the enterobactin genes fepB and entE. This mutation disrupted the structural gene for a previously identified 44-kilodalton protein and eliminated production of 2,3-dihydroxybenzoic acid, the catechol precursor of enterobactin. The complete nucleotide sequence of this gene was determined and compared with the sequences of other genes encoding chorismate-utilizing proteins. The similarities observed in these comparisons not only indicated that the locus is entC but also supported the premise that these enzymes constitute a family of related proteins sharing a common evolutionary origin. In addition, in this and the accompanying paper (M. S. Nahlik, T. J. Brickman, B. A. Ozenberger, and M. A. McIntosh, J. Bacteriol. 171:784-790, 1989), evidence is presented indicating that the entA product is potentially a secondary factor in the chorismate-to-isochorismate conversion and that the prototypic entC lesion (entC401) resides in the structural gene for the EntA protein. Finally, polarity effects from the insertion mutation in entC on downstream biosynthetic genes indicated that this locus is the promoter-proximal cistron in an ent operon comprising at least five genes. Appropriate regulatory signals upstream of entC suggest that this operon is regulated by iron through interaction with the Fur repressor protein.

Amino Acid Sequence↗
PubMed1992

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

Previous genetic analyses indicated that translational frameshifting in the--1 direction occurs within the run of six adenines in the sequence 5'-TTAAAAAACTC-3' at nucleotide positions 305-315 in IS 1, where the two out-of-phase reading frames insA and B'-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84-87. IS 1 mutants with a 1 bp insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84-87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-Lys-Lys-Leu. An IS 1 mutant with the DNA segment 5'-CTTAAAAACTC-3' at positions 305-315 carrying the termination codon TAA in the B'-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The beta-galactosidase activity specified by several IS 1-lacZ fusion plasmids, in which B'-insB is in-frame with lacZ, showed that the region 292-377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu-Lys-Lys-Leu encoded by the DNA segment 5'-TTAAAAAACTC-3', indicating that--1 frameshifting does occur within the run of adenines.

Adenine↗
PubMed1990

Identification and expression of the Escherichia coli fdhD and fdhE genes, which are involved in the formation of respiratory formate dehydrogenase.

The two closely linked fdhD and fdhE genes of Escherichia coli are required for the formation of active membrane-bound phenazine methosulfate-linked formate dehydrogenase (FDH-PMS). Both genes were isolated from a cosmid library. Restriction endonuclease analysis associated with Mu dII1734 insertion mutagenesis indicated that the two genes were separated by at least 4 kilobases and transcribed in opposite orientations. Initial experiments indicate that the region between the two genes seems not to be essential to FDH-PMS activity. fdhD and fdhE were expressed either in maxicells or from the T7 promoter-polymerase system. They were shown to encode proteins with approximate Mr 30,500 and 32,000, respectively. Both proteins appeared in the soluble fraction and were not recognized by an FDH-PMS-specific antiserum. Therefore, neither fdhD nor fdhE plays a structural role in the formation of FDH-PMS. Expression of a phi(fdhD-lacZ) operon fusion was decreased about threefold by aerobiosis but was indifferent to other effectors tested. It was unaffected by pfl, chlA, selA, and fnr mutations. Expression of a phi(fdhE-lacZ) operon fusion was slightly induced by nitrate. This induction, requiring the presence of functional chl and fnr alleles, was mediated via nitrate metabolism. Transcription of phi(fdhE-lacZ) fusion was fully dependent on wild-type sel alleles. This might suggest the participation of fdhE in the synthesis of the selenopolypeptide of FDH-PMS.

Cloning, Molecular↗
PubMed2003

Identification of genes required for synthesis of the adhesive holdfast in Caulobacter crescentus.

Adhesion to both abiotic and biotic surfaces by the gram-negative prothescate bacterium Caulobacter crescentus is mediated by a polar organelle called the "holdfast," which enables the bacterium to form stable monolayer biofilms. The holdfast, a complex polysaccharide composed in part of N-acetylglucosamine, localizes to the tip of the stalk (a thin cylindrical extension of the cell wall and membranes). We report here the isolation of adhesion mutants with transposon insertions in an uncharacterized gene cluster involved in holdfast biogenesis (hfs) as well as in previously identified polar development genes (podJ and pleC), and the holdfast attachment genes (hfa). Clean deletions of three of the four genes in the hfs gene cluster (hfsDAB) resulted in a severe holdfast biogenesis phenotype. These mutants do not bind to surfaces or to a fluorescently labeled lectin, specific for N-acetylglucosamine. Transmission electron microscopy indicated that the hfsDAB mutants fail to synthesize a holdfast at the stalk tip. The predicted hfs gene products have significant sequence similarity to proteins necessary for exopolysaccharide export in gram-negative bacteria. HfsA has sequence similarity to GumC from Xanthomonas campestris, which is involved in exopolysaccharide export in the periplasm. HfsD has sequence similarity to Wza from Escherichia coli, an outer membrane protein involved in secretion of polysaccharide through the outer membrane. HfsB is a novel protein involved in holdfast biogenesis. These data suggest that the hfs genes play an important role in holdfast export.

Adhesins, Bacterial↗
PubMed1987

Identification and characterization of genes determining receptor binding and pilus length of Escherichia coli type 1 pili.

We describe the identification and characterization of two genes and their gene products responsible for determining receptor binding and pilus length in type 1-piliated Escherichia coli. One gene, pilE, conferred the ability of piliated cells to agglutinate guinea pig erythrocytes. The other gene, pilF, determined pilus length, in that mutants having lesions in pilF had very long pili. The two genes were detected after Tn5 mutagenesis of a cloned segment of DNA that normally complemented a pilE lesion in the chromosome. Thus, lesions in pilE or pilF on the cloned segment resulted in mutants having the PilE- phenotype (piliated but unable to agglutinate erythrocytes). Introduction of the plasmid-encoded mutant alleles of pilE and pilF into the chromosome followed by electron microscopic examination of the mutants showed that only lesions in pilF conferred the striking increase in pilus length. Mutations in pilF could be complemented in trans by the original cloned segment to produce cells with parental-length pili. Minicell transcription and translation of the cloned pilE and pilF genes having representative Tn5 insertion mutations showed that the pilE gene product was a protein of ca. 31 kilodaltons and that the pilF gene product was a protein of ca. 18 kilodaltons. We believe that the pilF gene product may act as a competitive inhibitor of pilus polymerization. Thus, pilus length may be controlled by the ratio of pilin to pilF gene product present within the cell.

DNA Restriction Enzymes↗
PubMed1998

Identification of the contiguous Paracoccus denitrificans ccmF and ccmH genes: disruption of ccmF, encoding a putative transporter, results in formation of an unstable apocytochrome c and deficiency in siderophore production.

Apocytochrome C550 was detected in the periplasm of a new mutant of Paracoccus denitrificans, HN48, that is pleiotropically lacking c-type cytochromes, produces reduced levels of siderophores and carries a Tn5 insertion in the ccmF gene for which sequence data, along with that for the contiguous ccmH, are reported. A counterpart to the ccmF gene was found in an archaebacterium but could not be located in the yeast genome, whereas mitochondrial haem lyases in the latter were not present in an archaeobacterial or in eubacterial genomes. A topological analysis for CcmF is presented which indicates at least eleven transmembrane helices, suggesting a role as a transporter; evidence against the substrate being haem is presented but sequence similarity with Escherichia coli gamma-aminobutyric acid transporter was identified. Analysis by pulse-chase methodology has shown that, in this and another cytochrome-c-deficient mutant, the apo form of P. denitrificans cytochrome C550 is much less stable than the holo form, directly demonstrating the presence of a periplasmic degradation system in P. denitrificans that removes non-functional proteins. A variety of phenotypes are observed for P. denitrificans mutated in different ccm genes, thus indicating that the stability of the ccm gene products does not require assembly of a complex of all the Ccm proteins.

Amino Acid Sequence↗
PubMed2006

Identification of novel mutant PAX6 alleles in Indian cases of familial aniridia.

BACKGROUND: Haploinsufficiency at the PAX6 locus causes aniridia, a panocular eye condition characterized by iris hypoplasia and a variety of other anterior and posterior eye defects leading to poor vision. This study was performed to identify novel PAX6 mutations that lead to familial aniridia in Indian patients. METHODS: Genomic DNA was isolated from affected individuals (clinically diagnosed aniridia) from nine unrelated aniridic pedigrees, unaffected family members, and unrelated normal controls. The coding regions of PAX6 were amplified and subjected to single strand conformation polymorphism (SSCP) gel analysis, and direct cloning and sequencing. RESULTS: SSCP band shifts, indicative of DNA base pair mutations, were observed in five of these unrelated families. Four mutations were shown to be previously unreported insertion or deletions in PAX6, leading to frameshifts. These new mutations were c.1174delTG (in exon 10), c.710delC (exon 6), c.406delTT (exon 5) and c.393insTCAGC (exon 5). The other nonsense mutation, a transition (c.1080C>T) in exon 9, has been reported previously as a mutation hotspot for PAX6 in other ethnic pedigrees. All mutant alleles transmitted through aniridic individuals in each family. CONCLUSION: These new deletions and an insertion create frameshifts, which are predicted to introduce premature termination codons into the PAX6 reading frame. The genetic alterations carried by affected individuals are predicted to lead to loss-of-function mutations that would segregate in an autosomal dominant manner to subsequent generations. This is the first report of the 'hotspot' c.1080C>T transition from Indian families.

Alleles↗
PubMed1995

Detection of Rhizobium meliloti cells in field soil and nodules by polymerase chain reaction.

A genetically marked Rhizobium meliloti strain, R692, was prepared by insertion of a 1.7-kb DNA segment from Tn903 between the nifHDK and fixABC genes in the nod megaplasmid. This DNA was used as a marker, detectable by polymerase chain reaction (PCR), for the specific identification of bacteria in soil samples and alfalfa nodules. This detection technique was tested by applying different titres of the marked strain to field plots seeded with alfalfa. Samples of soil and nodules were assayed for the presence of the marker DNA fragment by PCR using primers specific to the marker sequence. The experiments revealed that the bacteria could be detected directly in soil containing about 10(3)-10(4) bacteria/g, but greater sensitivity was prevented by potent PCR inhibitors present in the samples. The titre of the bacteria in the soil decreased rapidly after inoculation, dropping about 10-fold per week. Tests of vertical location of the bacteria in soil cores showed that the bacteria were initially dispersed to a depth of 18 cm, and subsequently retained viability in the top 2-8 cm. As few as 10 marked R. meliloti per gram of soil resulted in its establishment at detectable levels in nodules. Application of about 10(4)-10(5) bacteria/g soil was sufficient to give the maximum number of nodules per plant and resulted in 70-90% occupancy by the marked strain. Limited movement of the inoculant was detected by analysis of nodules from plants adjacent to the sites where the bacteria were applied, probably by movement in water. The experiments demonstrated the advantages of PCR for the monitoring of marked microorganisms in the environment.

Base Sequence↗
PubMed1994

Identification and characterization of a Salmonella typhimurium oxygen-regulated gene required for bacterial internalization.

Growth of Salmonella typhimurium in a low-oxygen environment induces the ability of these bacteria to enter mammalian cells. We have carried out a search for invasion genes that are expressed under low-oxygen conditions by using Tn5lacZY transcriptional fusions. Several noninvasive oxygen-regulated lacZY insertion strains have been identified. The invasion defect in one of these noninvasive S. typhimurium strains, BJ66, has been complemented by introduction of a cosmid (pBDJ125) from an S. typhimurium SL1344 gene bank. A 1.9-kb EcoRV DNA fragment subcloned from this cosmid, containing a single open reading frame (orgA), restores the ability of BJ66 to invade mammalian cells. Comparative searches of the GenBank and EMBL sequence data banks with the nucleotide sequence of the gene and deduced amino acid sequence of the protein reveal no significant similarities. Interestingly, hybridization of an orgA gene probe with a P22 chromosomal mapping library demonstrated that the orgA gene maps to a region on the chromosome between 57.5 and 60 min where other Salmonella invasion genes have been mapped. Other enteroinvasive bacteria (Shigella flexneri, Escherichia coli, Yersinia spp., and Listeria monocytogenes) lack sequences which cross hybridize to the probe. We have compared the virulence of S. typhimurium SL1344 and an isogenic orgA mutant in a mouse model of typhoid fever. The orgA mutant was as virulent as the wild-type strain was when inoculated intraperitoneally but is significantly reduced (> 60-fold) in its ability to cause disease by an oral route of infection.

Amino Acid Sequence↗
PubMed1987

Identification of uhp polypeptides and evidence for their role in exogenous induction of the sugar phosphate transport system of Escherichia coli K-12.

Cells of Escherichia coli possess a transport system that catalyzes the accumulation, in unaltered form, of a variety of sugar phosphates. Induction of the transport activity occurs in response to external glucose 6-phosphate and does not require detectable entry of this inducer. To define the genes that encode the Uhp transport system and those that mediate its exogenous induction, transposon insertions were isolated and mapped within a 6.5-kilobase HindIII-BamHI fragment that carries the entire uhp region. The transposon insertions were transferred by homologous recombination onto the chromosome to test their effect on Uhp expression when all genes were present in single copy number. The complementation behavior of plasmids carrying the insertions or subcloned fragments of the region was compared with their polypeptide coding capacity in maxicells. These studies defined three uhp regulatory genes (uhpABC), all of which are necessary for expression of the uhpT gene, which encodes the transporter. The products of uhpB and uhpC are not required when uhpA is present on a multicopy plasmid. The four genes, uhpA, uhpB, uhpC, and uhpT, are transcribed in the same direction, and their products have apparent molecular weights of 25,000, 48,000, 20,000, and 38,000, respectively. The UhpB and UhpT polypeptides are associated with the membrane fraction. These results led to a model of regulation in which the UhpB and UhpC regulatory proteins prevent the ability of UhpA to activate transcription of the uhpT gene under noninducing conditions.

Bacterial Proteins↗
PubMed1992

Identification of a Salmonella typhimurium invasion locus by selection for hyperinvasive mutants.

Salmonella typhimurium penetrate intestinal epithelial cells during infection. In vitro studies reveal that the availability of oxygen during bacterial growth decreases their capacity to adhere to and enter cultured epithelial cells. To identify S. typhimurium genes involved in epithelial cell entry, mutants were selected that entered HEp-2 cells when grown under repressing, aerobic culture conditions. Two types of transposons were used to generate bacterial mutations--transposons that disrupt genes (Tn10 and Tn5) and one transposon (Tn5B50) that, in addition to disrupting genes, can cause constitutive expression of genes from the neo promoter at one end of the transposon. Three classes of mutations were found that increased the ability of aerobically grown S. typhimurium to enter HEp-2 cells. One class of mutations disrupts the che operons and results in a nonchemotactic phenotype. The second class of mutations revealed that defects in rho, which encodes an essential transcription termination factor, result in hyperinvasiveness. The third class of mutations was obtained only from mutagenesis with Tn5B50, suggesting that their increased invasiveness is due to constitutive expression of a gene(s) from the exogenous neo promoter. Analysis of this third class of mutations identified a S. typhimurium locus hil (hyperinvasion locus), which is essential for bacterial entry into epithelial cells. The results suggest that hil encodes an invasion factor or an activator of invasion factor expression. hil maps between srl and mutS near minute 59.5 of the S. typhimurium chromosome, a region adjacent to other loci that have been identified as required for S. typhimurium invasiveness and virulence.

Anaerobiosis↗
PubMed1994

Identification of the region that determines the specificity of binding of the transposases encoded by Tn3 and gamma delta to the terminal inverted repeat sequences.

To analyze the region that determines the specificity of binding of the Tn3 transposase to the terminal inverted repeat sequences (IR), we first determined the nucleotide sequence of a Tn3-family transposon, gamma delta, which is supposed to encode a transposase similar to that of Tn3. gamma delta was 5981 bp in length and contained three coding frames: Two were the genes, tnpA and tnpR, encoding transposase (1002 amino acids) and resolvase/repressor (183 amino acids), respectively, and the third, named tnpX, encoding a protein (698 amino acids) of unknown function but containing two NTP-binding motifs. Utilizing the tnpA sequence, we then constructed a series of Tn3-gamma delta hybrid genes encoding chimeric proteins in the N-terminal segments of the transposases (amino acid position 1 to 242 of Tn3 or 1' to 243' of gamma delta), which has been previously shown to be responsible for specific binding of transposase to IR sequences in Tn3. Examination of their DNA-binding activities revealed that the subsegment of the N-terminus from amino acid position 1 to 109 determines the specificity of binding to the IR sequences. The third coding frame found in gamma delta, tnpX, is located downstream of tnpR and is expressed from the tnpR promoter in the absence of the tnpR gene product, resolvase/repressor, to produce a protein that inhibits the growth of the host cells. Possible roles of this protein are discussed.

Amino Acid Sequence↗
PubMed2003

A newly discovered Bacteroides conjugative transposon, CTnGERM1, contains genes also found in gram-positive bacteria.

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.

Bacteroides↗