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PubMed2006

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli.

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.

Amino Acid Sequence↗
PubMed1998

SOX10 mutations in patients with Waardenburg-Hirschsprung disease.

Waardenburg syndrome (WS; deafness with pigmentary abnormalities) and Hirschsprung's disease (HSCR; aganglionic megacolon) are congenital disorders caused by defective function of the embryonic neural crest. WS and HSCR are associated in patients with Waardenburg-Shah syndrome (WS4), whose symptoms are reminiscent of the white coat-spotting and aganglionic megacolon displayed by the mouse mutants Dom (Dominant megacolon), piebald-lethal (sl) and lethal spotting (ls). The sl and ls phenotypes are caused by mutations in the genes encoding the Endothelin-B receptor (Ednrb) and Endothelin 3 (Edn3), respectively. The identification of Sox10 as the gene mutated in Dom mice (B.H. et al., manuscript submitted) prompted us to analyse the role of its human homologue SOX10 in neural crest defects. Here we show that patients from four families with WS4 have mutations in SOX10, whereas no mutation could be detected in patients with HSCR alone. These mutations are likely to result in haploinsufficiency of the SOX10 product. Our findings further define the locus heterogeneity of Waardenburg-Hirschsprung syndromes, and point to an essential role of SOX10 in the development of two neural crest-derived human cell lineages.

Amino Acid Sequence↗
PubMed1989

Characterization of five genes in the upper-pathway operon of TOL plasmid pWW0 from Pseudomonas putida and identification of the gene products.

The upper operon of the TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. The genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in Escherichia coli maxicells. This analysis showed that the upper operon contains at least five genes in the order of xylC-xylM-xylA-xylB-xylN. Between the promoter of the operon and xylC, there is a 1.7-kilobase-long space of DNA in which no gene function was identified. In contrast, most of the DNA between xylC and xylN consists of coding sequences. The xylC gene encodes the 57-kilodalton benzaldehyde dehydrogenase. The xylM and xylA genes encode 35- and 40-kilodalton polypeptides, respectively, which were shown by genetic complementation tests to be subunits of xylene oxygenase. The structural gene for benzyl alcohol dehydrogenase, xylB, encodes a 40-kilodalton polypeptide. The last gene of this operon is xylN, which synthesizes a 52-kilodalton polypeptide of unknown function.

DNA Transposable Elements↗
PubMedDate not supplied

Isolation of Mycobacterium bovis from baboons, leopards and a sea-lion.

This study reports on two series of cases of Mycobacterium bovis infection in zoo animals. The first was in a captive population of baboons (Papio hamadryas) and the second in a mixed group of wild mammals, including four leopards (Panthera uncia and Panthera pardus) and a sea-lion (Otaria byrona). The isolation and identification of strains of M. bovis confirmed the presence of M. bovis infections in both zoos. The epidemiological study using genetic markers such as the IS6110-based DNA fingerprinting system made it possible to differentiate between M. bovis strains. The M. bovis strains isolated from baboons were shown to contain a single IS6110 copy, as usually do cattle isolates, whereas the M. bovis strains isolated from the other exotic animals presented multiple copies. This finding suggests that the origin of the contamination for the baboons in zoo A could be related to cattle. The origin of the contamination for the leopards and sea-lion in zoo B is more difficult to determine. In conclusion, the authors suggest some recommendations for avoiding outbreaks of tuberculosis infections in zoos.

Animals↗
PubMed1989

A specific class of IS10 transposase mutants are blocked for target site interactions and promote formation of an excised transposon fragment.

We report the identification and characterization of a class of IS10 transposase mutants that carry out only some of the steps required for transposition. These mutants were identified among transposition-defective mutants as a specific subclass that retains the wild-type ability to induce SOS functions in the presence of transposon ends. Mutants of this class successfully promote excision of the element from its donor site, but do not promote transfer of the transposon sequences to a target site. SOS induction presumably results from the degradation of the donor site. Uniquely among transposition-defective mutants, SOS+ Tnsp- mutants promote the formation of a new product, the excised transposon fragment (ETF), which consists of the transposon excised from the original donor molecule by double-strand breaks at the transposon ends. SOS+ Tnsp- mutants identified thus far define two patches of amino acids that might correspond to regions of different function. A single additional mutation maps within a region that is highly conserved among IS element transposases. The existence of SOS+ Tnsp- mutants and the structure of the ETF provide strong support for the previously proposed nonreplicative model of Tn10/IS10 transposition.

Alleles↗
PubMed1987

Identification of a new insertion element, similar to gram-negative IS26, on the lactose plasmid of Streptococcus lactis ML3.

In Streptococcus lactis ML3, the lactose plasmid (pSK08) forms cointegrates with a conjugal plasmid (pRS01). It has been proposed that cointegration is mediated by insertion sequences (IS) present on pSK08 (D. G. Anderson and L.L. McKay, J. Bacteriol. 158:954-962, 1984). We examined the junction regions of the cointegrate pPW2 and the corresponding regions of pSK08 (donor) and pRS01 (target) and identified a new IS element on pSK08 (ISS1S) which was involved in and duplicated during formation of pPW2. ISS1S was 808 base pairs (bp) in size, had 18-bp inverted repeats (GGTTCTGTTGCAAAGTTT) at its ends, contained a single long open reading frame encoding a putative protein of 226 amino acids, and generated 8-bp direct repeats of target DNA during cointegrate formation. An iso-IS element, ISS1T, which is duplicated in some other cointegrate plasmids, was also found on pSK08. ISS1T was also 808 bp in size and was identical to ISS1S in sequence except for 4 bp, none of which altered the inverted repeats or amino acid sequence of the open reading frame. Comparison of ISS1 with gram-negative IS26 revealed strong homologies in size (820 bp), sequence of inverted repeats (GGCACTGTTGCAAA), size of direct repeats generated after cointegration (8 bp), and number, size, and amino acid sequence (44.5% identical) of the open reading of frame.

Amino Acid Sequence↗
PubMed2006

Identification of genes involved in swarming motility using a Pseudomonas aeruginosa PAO1 mini-Tn5-lux mutant library.

During a screening of a mini-Tn5-luxCDABE transposon mutant library of Pseudomonas aeruginosa PAO1 for alterations in swarming motility, 36 mutants were identified with Tn5 insertions in genes for the synthesis or function of flagellin and type IV pilus, in genes for the Xcp-related type II secretion system, and in regulatory, metabolic, chemosensory, and hypothetical genes with unknown functions. These mutants were differentially affected in swimming and twitching motility but in most cases had only a minor additional motility defect. Our data provide evidence that swarming is a more complex type of motility, since it is influenced by a large number of different genes in P. aeruginosa. Conversely, many of the swarming-negative mutants also showed an impairment in biofilm formation, indicating a strong relationship between these types of growth states.

Bacterial Proteins↗
PubMed2003

Molecular signatures in protein sequences that are characteristic of cyanobacteria and plastid homologues.

Fourteen conserved indels (i.e. inserts or deletions) have been identified in 10 widely distributed proteins that appear to be characteristic of cyanobacterial species and are not found in any other group of bacteria. These signatures include three inserts of 6, 7 and 28 aa in the DNA helicase II (UvrD) protein, an 18-21 aa insert in DNA polymerase I, a 14 aa insert in the enzyme ADP-glucose pyrophosphorylase, a 3 aa insert in the FtsH protein, an 11-13 aa insert in phytoene synthase, a 5 aa insert in elongation factor-Tu, two deletions of 2 and 7 aa in ribosomal S1 protein, a 2 aa insert in the SecA protein, a 1 aa deletion and a 6 aa insert in the enzyme inosine-5'-monophosphate dehydrogenase and a 1 aa deletion in the major sigma factor. These signatures, which are flanked by conserved regions, provide molecular markers for distinguishing cyanobacterial taxa from all other bacteria and they should prove helpful in the identification of cyanobacterial species, simply on the basis of the presence or absence of these markers in the corresponding proteins. The signatures in six of these proteins (SecA, elongation factor-Tu, ADP-glucose pyrophosphorylase, phytoene synthase, FtsH and ribosomal S1 protein) are also commonly present in plastid homologues from plants and algae (chlorophytes, chromophytes and rhodophytes), indicating their specific relationship to cyanobacteria and supporting their endosymbiotic origin from these bacteria. In phylogenetic trees based on a number of these proteins (SecA, UvrD, DNA polymerase I, elongation factor-Tu) that were investigated, the available cyanobacterial homologues grouped together with high affinity (>95 % bootstrap value), supporting the view that the cyanobacterial phylum is monophyletic and that the identified signatures were introduced in a common ancestor of this group.

Adenosine Triphosphatases↗
PubMed1998

Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks.

From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified. The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC. In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC. Transduction of most of the transposon mutations to a fresh S. typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S. typhimurium F98 supported the role in colonization of all but the pts locus. The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested. All but the ptsC and rfaY mutants were attenuated for virulence. A number of other phenotypes associated with some of the mutations are described.

Animals↗
PubMed1984

Identification of the sporulation gene spoOA product of Bacillus subtilis.

A 2.4-kilobase fragment of the Bacillus subtilis chromosome containing the wild-type spoOA gene derived from the phi 105dspoOA+-Bc-1 transducing phage was cloned onto plasmid pBR322 in Escherichia coli. A recombinant plasmid harboring the mutant spoOA12 allele on the 2.4-kilobase insert was also constructed from the phi 105dspoOA12-1 phage DNA and pBR322. Protein products synthesized in response to plasmid DNA in a DNA-directed cell-free system derived from E. coli were analyzed by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A protein of approximately 27,500 daltons synthesized with the recombinant plasmid DNA harboring the wild-type spoOA gene as template was not formed with the recombinant plasmid DNA harboring the spoOA12 allele. Since the spoOA12 mutation is a nonsense mutation, we conclude that the 27.5-kilodalton protein is the product of the spoOA gene.

Alleles↗
PubMed2001

Identification of chromosome inheritance modifiers in Drosophila melanogaster.

Faithful chromosome inheritance is a fundamental biological activity and errors contribute to birth defects and cancer progression. We have performed a P-element screen in Drosophila melanogaster with the aim of identifying novel candidate genes involved in inheritance. We used a "sensitized" minichromosome substrate (J21A) to screen approximately 3,000 new P-element lines for dominant effects on chromosome inheritance and recovered 78 Sensitized chromosome inheritance modifiers (Scim). Of these, 69 decreased minichromosome inheritance while 9 increased minichromosome inheritance. Fourteen mutations are lethal or semilethal when homozygous and all exhibit dramatic mitotic defects. Inverse PCR combined with genomic analyses identified P insertions within or close to genes with previously described inheritance functions, including wings apart-like (wapl), centrosomin (cnn), and pavarotti (pav). Further, lethal insertions in replication factor complex 4 (rfc4) and GTPase-activating protein 1 (Gap1) exhibit specific mitotic chromosome defects, discovering previously unknown roles for these proteins in chromosome inheritance. The majority of the lines represent mutations in previously uncharacterized loci, many of which have human homologs, and we anticipate that this collection will provide a rich source of mutations in new genes required for chromosome inheritance in metazoans.

Animals↗
PubMed1999

Identification of a novel nutrient-deprivation-induced Sinorhizobium meliloti gene (hmgA) involved in the degradation of tyrosine.

Sinorhizobium meliloti strain N4 carries a Tn5luxAB insertion in a gene which is induced by nitrogen and carbon deprivation as well as in the presence of tyrosine. The Tn5luxAB-tagged locus was found to share significant similarity with the human hmgA gene and the corresponding Aspergillus nidulans gene, encoding the enzyme homogentisate dioxygenase, which is involved in the degradation of tyrosine. Extended DNA sequence analysis of the tagged locus revealed the presence of several ORFs, including one encoding a polypeptide sharing a high degree of similarity with human and fungal maleylacetoacetate isomerases. Strain N4 was found to be unable to use tyrosine as carbon source, to lack homogentisate dioxygenase activity, to produce a melanin-like pigment and to be affected in stationary-phase survival. This is believed to be the first report of a hmgA-homologous gene in bacteria.

Amino Acid Sequence↗
PubMed2004

Inactivation of pycA, encoding pyruvate carboxylase activity, increases poly-beta-hydroxybutyrate accumulation in Azotobacter vinelandii on solid medium.

Strain AJ1678, an Azotobacter vinelandii mutant overproducing the storage polymer poly-beta-hydroxybutyrate (PHB) in solid but not liquid complex medium with sucrose, was isolated after mini-Tn5 mutagenesis of strain UW136. Cloning and nucleotide sequencing of the affected locus led to identification of pycA, encoding a protein with high identity to the biotin carboxylase subunit of pyruvate carboxylase enzyme (PYC). A gene ( pycB) whose product is similar to the biotin-carrying subunit of PYC is present immediately downstream from pycA. An assay of pyruvate carboxylase activity and an avidin-blot analysis confirmed that pycA and pycB encode the two subunits of this enzyme. In many organisms, PYC catalyzes ATP-dependent carboxylation of pyruvate to generate oxaloacetate and is responsible for replenishing oxaloacetate for continued operation of the tricarboxylic acid cycle. We propose that the pycA mutation causes a slow-down in the TCA cycle activity due to a low oxaloacetate concentration, resulting in a higher availability of acetyl-CoA for the synthesis of poly-beta-hydroxybutyrate.

Acetyl Coenzyme A↗
PubMed1990

Insertion of the fusion gene from Newcastle disease virus into a non-essential region in the terminal repeats of fowlpox virus and demonstration of protective immunity induced by the recombinant.

In this paper we report on the identification of non-essential genes in the terminal repeats of the avipox-virus fowlpox virus and the use of these as insertion sites in a vector system. Foreign genes inserted into these sites are shown to be present in two copies in the resultant recombinant virus. To test the potential use of this vector as a live vaccine the fusion gene of Newcastle disease virus has been inserted into a vaccine strain of fowlpox virus and inoculated into chickens. The experiments demonstrate the ability of the recombinant to protect chickens against challenge by a virulent strain of Newcastle disease virus and to elicit the formation of an anti-fusion protein antibody.

Animals↗
PubMed1995

A transposon-like sequence adjacent to the AccI restriction-modification operon.

We have cloned and sequenced the accIRM genes from Weeksella zoohelcum (the original identification of this strain as Acinetobacter calcoaceticus was incorrect). Our sequence differs in the coding regions from a previously published sequence by the addition of three nucleotides near the 3' end of the DNA methyltransferase-encoding gene (accIM). We have sequenced approx. 3 kb beyond this operon. Two genes were found, convergently transcribed with the R-M operon. The first of these genes encodes a protein which shows significant similarity to the recombinases of the phage integrase family. The W. zoohelcum recombinase may function as a transposon resolvase, as in Tn4430. The recombinase-encoding gene is followed by a putative transposase (Tnp), which is in turn followed by a terminator which is predicted to be Rho-dependent for the recombinase-Tnp operon and Rho-independent for the convergent R-M operon. Since the G + C content of the two operons is notably different, it is possible that the terminator is at the extremity of the mobile element and serves to protect it from incoming transcription.

Amino Acid Sequence↗
PubMed1991

Identification of a new operon involved in Listeria monocytogenes virulence: its first gene encodes a protein homologous to bacterial metalloproteases.

The region flanking the transposon in a Tn1545-induced lecithinase-negative mutant of Listeria monocytogenes EGD was cloned and sequenced. The transposon had inserted in ORF D, the open reading frame previously identified downstream from hlyA, the gene encoding listeriolysin O. The complete sequence of ORF D from strain EGD has been determined as well as that of two other strains: LO28, a clinical isolate; and LM8, an epidemic strain. ORF D is 1,533 bp long and encodes a protein highly homologous to metalloproteases of bacilli, Serratia sp., Legionella pneumophila, and Pseudomonas aeruginosa. It was renamed prtA. Northern RNA blot analysis indicated that prtA is the first gene of a 6-kb operon, suggesting that the lecithinase-negative phenotype of the mutant might be due to a polar effect of the transposon insertion.

Amino Acid Sequence↗
PubMed1985

Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens.

Analyses were made of the host-dependent-variation (hdv) locus of the virulence (vir) region of the pTiC58 plasmid of Agrobacterium tumefaciens. The hdv locus is comprised of at least four genes that encode polypeptides of 13, 15, 29, and 28 kDa. Insertion of transposon Tn5 in the first gene abolishes the expression of all four genes in vitro and in vivo. Nucleotide sequence analysis of the hdv locus revealed four open reading frames tandemly arranged with spacer sequences having no promoter-like sequences and lacking the ability to bind A. tumefaciens RNA polymerase. These studies suggest that the hdv locus is comprised of at least four genes arranged in an operon in the vir region. The protein products of these genes are likely to function in some aspect of the host-range determination of A. tumefaciens.

Amino Acid Sequence↗
PubMed1992

Identification of a streptomycin resistance gene and a partial Tn3 transposon coding for a beta-lactamase in a periodontal strain of Eikenella corrodens.

The beta-lactamase gene from a periodontal strain of Eikenella corrodens, resistant to penicillins and streptomycin, was inserted into pBGS9 and transformed into Escherichia coli DH5 alpha. A 4.7-kb insert of pJML1, one of the transformants, was partially sequenced and found to contain the right section of transposon Tn3 from the middle of the TnpR resolvase gene to the right inverted repeat RI(R), including the TEM-1 gene. Sequences identical to RSF1010 were found on either side of the Tn3 sequence. pJML1 also contained a streptomycin resistance gene, probably identical to that of RSF1010. A portion of the pJML1 insert was not homologous to either Tn3 or RSF1010 but was homologous to the chromosomal DNA of E. corrodens ATCC 23834. It is assumed that the insert of pJML1 was derived from the chromosomal DNA of E. corrodens EC-38.

DNA Transposable Elements↗