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Biofilm formation in vitro and virulence in vivo of mutants of Klebsiella pneumoniae.

One of the early stages of Klebsiella pneumoniae airway infections may involve biofilm formation. Bacterial biofilm formation is frequently investigated using in vitro techniques that facilitate identification and analysis of individual genes. We investigated the correlation between K. pneumoniae biofilm formation in vitro and ability to cause infection in vivo following construction of a bank of mini-Tn5 mutants.

Animals↗

Complex transmission dynamics of clonally related virulent Mycobacterium tuberculosis associated with barhopping by predominantly human immunodeficiency virus-positive gay men.

Limited data suggest that measures to reduce tuberculosis transmission should be based on locations rather than on personal contacts. Molecular epidemiologic methods (analysis of IS6110 patterns, spoligotypes, variable numbers of tandem DNA repeats, and automated DNA sequence data) identified a cohort of 48 persons who were infected with progeny of the same Mycobacterium tuberculosis strain. Epidemiologic investigation documented that a large proportion of the patients were gay white human immunodeficiency virus-positive men. Most practiced barhopping, an activity that involved patronizing many bars in the same neighborhood each night. Few subjects were directly linked to more than 1 or 2 other persons by conventional investigation methods, which shows that the transmission dynamics were unusually complex compared with most previously described episodes of strain spread. The data support the concept that identification of locations where pathogen dissemination likely occurs may provide additional strategies for targeted tuberculosis control.

Alcohol Drinking↗

Sequence and TnphoA analysis of a Mycoplasma hyorhinis protein with membrane export function.

Proteins translocated across the single plasma membrane of mycoplasmas (class Mollicutes) represent important components likely to affect several interactions of these wall-less microbes with their respective hosts. However, identification and functional analysis of such proteins is hampered by the lack of mutational systems in mycoplasmas and by a perceived limitation in translating recombinant mycoplasma genes containing UGA (Trp) codons in other eubacteria. Here we directly analyze a gene encoding a Mycoplasma hyorhinis protein capable of promoting its membrane translocation. It was initially detected by screening a recombinant phage genomic library with antibody from a host with M. hyorhinis-induced arthritis and was localized by Tn5 and deletion mutations affecting expression of antigenic translational products. Sequence analysis of the isolated gene predicted a hydrophilic protein, P101, containing three UGA codons and a putative signal peptide with an uncharacteristic cluster of positively charged amino acids near its C terminus. Nevertheless, lambda::TnphoA transposon mutagenesis of an Escherichia coli plasmid bearing the p101 gene resulted in p101::TnphoA fusions expressing products that could translocate as much as 48 kDa of the P101 sequence (up to the first UGA codon) across the E. coli plasma membrane. Fusion proteins containing mature P101 sequences expressed mycoplasma epitopes and were found by cell fractionation and detergent phase partitioning to be integral membrane proteins in E. coli, suggesting a lack of signal peptide cleavage in this system. Importantly, identification of P101 by direct analysis of its export function relied neither on prior identification of the mycoplasmal product nor on complete expression of the product from the cloned mycoplasma gene.

Amino Acid Sequence↗

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome.

BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.

4-Butyrolactone↗

Insertional mutagenesis in C. elegans using the Drosophila transposon Mos1: a method for the rapid identification of mutated genes.

One benefit of the nematode Caenorhabditis elegans as a model system is the ease to conduct forward genetic screens and to isolate mutants with phenotypes of interest. However, identifying the mutated genes requires positional cloning, which can be laborious and time consuming. Insertional mutagenesis with a heterologous transposon bypasses the mapping steps and expedites the process of identifying the mutated genes. The Drosophila transposon Mos1 can be mobilized in the C. elegans germline to cause mutations. Mutagenic insertions are subsequently localized within the genome using inverse polymerase chain reaction. The mutagenicity of this technique is roughly one order of magnitude lower than chemical mutagens. However, the molecular identification of the mutated genes is extremely rapid. Therefore, before using Mos1-mediated mutagenesis, one must evaluate the trade-off between time spent screening for mutants vs time spent mapping and rescuing a mutation.

Animals↗

[Biotinylated DNA probe for detection of streptotricin acetylation genes].

The biotin-labelled DNA probe for identification of functioning and silent genes for streptotricin acetylation has been constructed. The probe is homologous to sat1 gene of the movable genetic element Tn1825. The simplified modification of the hybridization technique using the biotin-labelled DNA probe is described.

Acetylation↗

The 51,409-bp R-plasmid pTP10 from the multiresistant clinical isolate Corynebacterium striatum M82B is composed of DNA segments initially identified in soil bacteria and in plant, animal, and human pathogens.

The 51,409-bp DNA sequence of the multiresistance plasmid pTP10 from the gram-positive opportunistic human pathogen Corynebacterium striatum M82B has been determined. Fully automated genome interpretation led to the identification of 47 ORFs. Analysis of the genetic organization of pTP10 suggests that the plasmid is composed of eight DNA segments, the boundaries of which are represented by transposons and insertion sequences. The DNA segments of pTP10 are highly similar to (1) a plasmid-encoded erythromycin resistance region from the human pathogen Corynebacterium diphtheriae; (2) a chromosomal DNA region from Mycobacterium tuberculosis; (3) a plasmid-encoded chloramphenicol resistance region from the soil bacterium Corynebacterium glutamicum; (4) transposable elements from phytopathogenic gram-negative Pseudomonas, Xanthomonas and Erwinia species; and (5) a plasmid-encoded aminoglycoside resistance region from the gram-negative fish pathogen Pasteurella piscicida. The complete DNA sequence of pTP10 provides genetic information regarding the mechanisms of resistance to 16 antimicrobial agents that belong to six structural classes. In addition, the mosaic structure of pTP10 represents the evolutionary consolidation into a single plasmid molecule of antimicrobial resistances from microorganisms found in different habitats by means of mobile elements, resulting in the generation of a multiresistant bacterium that can infect humans.

Animals↗

Modular bacterial artificial chromosome vectors for transfer of large inserts into mammalian cells.

To facilitate the use of large-insert bacterial clones for functional analysis, we have constructed new bacterial artificial chromosome vectors, pPAC4 and pBACe4. These vectors contain two genetic elements that enable stable maintenance of the clones in mammalian cells: (1) The Epstein-Barr virus replicon, oriP, is included to ensure stable episomal propagation of the large insert clones upon transfection into mammalian cells. (2) The blasticidin deaminase gene is placed in a eukaryotic expression cassette to enable selection for the desired mammalian clones by using the nucleoside antibiotic blasticidin. Sequences important to select for loxP-specific genome targeting in mammalian chromosomes are also present. In addition, we demonstrate that the attTn7 sequence present on the vectors permits specific addition of selected features to the library clones. Unique sites have also been included in the vector to enable linearization of the large-insert clones, e. g., for optical mapping studies. The pPAC4 vector has been used to generate libraries from the human, mouse, and rat genomes. We believe that clones from these libraries would serve as an important reagent in functional experiments, including the identification or validation of candidate disease genes, by transferring a particular clone containing the relevant wildtype gene into mutant cells or transgenic or knock-out animals.

Animals↗

Cloning and characterisation of nifLA regulatory mutations from Klebsiella pneumoniae.

A total of nine regulatory mutations in the nifLA operon of Klebsiella pneumoniae were cloned in the high copy-number plasmid vector pACYC184. The regulatory phenotypes of the resultant clones were then correlated with their restriction maps and their ability to synthesise nifL and nifA polypeptides in vivo. One mutation, nifL2401, was identified as a 400 bp. deletion within the nifL gene. This mutation was non-polar and caused a Nif+ phenotype which showed escape from repression by oxygen and low levels of fixed nitrogen. Identification of this deletion allows the first definitive allocation of a mutation with this phenotype to the nifL gene and provides further evidence for the role of the nifL gene product in nif-specific repression.

Chromosome Mapping↗

Physical mapping and functional assignment of the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides.

The bacteriochlorophyll of the purple photosynthetic bacterium Rhodobacter sphaeroides is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that R. sphaeroides is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An R. sphaeroides transposon mutant in this gene, bchP, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a bchP gene product.

Amino Acid Sequence↗

Assessment of genetic markers for species differentiation within the Mycobacterium tuberculosis complex.

It is important to correctly identify species within the Mycobacterium tuberculosis complex because of the zoonotic implications of bovine tuberculosis, especially in developing countries. We assessed the use of various genetic markers for species-specific identification of mycobacteria from the M. tuberculosis complex. A multiplex PCR designed for detection of the mtp40 and IS1081 elements was optimized and evaluated in 339 mycobacterial strains from different animal and geographic origins. The host range of the IS6110, MPB70, and 16S rRNA genes was also studied by PCR in all the strains. Finally, the usefulness of the genetic markers was compared by an immunoperoxidase test for specific identification of Mycobacterium bovis strains. The mtp40 sequence was detected in 87 of the 91 strains of M. tuberculosis and in 9 of the 11 Mycobacterium africanum strains but not in any of the M. bovis or Mycobacterium microti strains, indicating that the mtp40 element was also found in all of the M. tuberculosis complex strains isolated from seals. This organism is considered to be a true seal pathogen, but its origin is essentially unknown. The finding of the mtp40 element in the strains from seals suggests a closer relationship of these strains with a human origin than to an animal origin. The mtp40 element was not found in any other mycobacterial species included in the study. As a result of this study, we suggest that biochemical tests or alternate genetic markers are still needed to differentiate M. tuberculosis from M. africanum when these species coexist as causative agents of tuberculosis. The immunoperoxidase test worked well for the identification of M. bovis strains. We also report, for the first time, PCR amplification of the repetitive element IS6110 in an isolate of Mycobacterium ulcerans and an isolate of Mycobacterium gilvum, which emphasizes the need for further investigation of the host range of this sequence.

Animals↗

Transforming science: cancer gene identification.

Methods for cancer gene discovery include identification of viral oncogenes, identification of genes associated with recurrent chromosomal aberrations, and screens for genes capable of the transformation of cells in culture. In recent years, the completed genome sequence of human and model organisms has markedly enhanced cancer gene identification. Whole genome, high-throughput screens have been facilitated by the advent of new technologies such as murine leukemia virus-based mutagenesis, Sleeping Beauty-based mutagenesis, RNA interference, exon re-sequencing, and high-resolution methods for detecting chromosomal amplifications and deletions; these, in turn, have led to the identification of novel tumor suppressors and oncogenes. The identification of genes that are altered by mutation or expression and which are directly involved in tumor initiation and maintenance will be instrumental for understanding cancer phenotypic variation and for identifying crucial therapeutic targets.

Animals↗

Iron regulation of siderophore biosynthesis and transport in Pseudomonas putida WCS358: involvement of a transcriptional activator and of the Fur protein.

Pseudobactin 358 is the yellow-green fluorescent siderophore produced by Pseudomonas putida WCS358 in conditions of iron limitation. The genes encoding for siderophore biosynthesis are iron-regulated at the transcriptional level. Previous work has shown that a positive regulator, PfrA, is absolutely required for the activation under iron-limiting conditions of pseudobactin 358 biosynthesis. In this study we identified a set of Tn5 insertion mutants of strain WCS358 which lost the ability to activate an iron-regulated siderophore promoter. These mutants no longer produced pseudobactin 358. Molecular analysis revealed that they carried a Tn5 insertion in a gene, designated pfrl (Pseudomonas ferric regulator), which codes for a protein (Pfrl) of 19.5 kDa. Pfrl contains a putative helix-turn-helix motif typical of DNA-binding proteins and has homology to two DNA-binding transcriptional activators, Fecl from Escherichia coli and Pupl from P. putida. The proposed role of Pfrl in strain WCS358 is an activator protein regulating pseudobactin 358 biosynthesis under iron limitation. The pfrl promoter region contains a sequence which displays high identity to the Fur-box consensus. This 19 bp consensus sequence is recognized by Fur, an iron-binding repressor protein found in many different bacteria. The E. coli Fur protein can bind to the pfrl promoter region, indicating that this activator gene is likely to be iron-regulated by Fur. We also report the identification and characterization of the P. putida WCS358 fur gene. The Fur protein of strain WCS358 is structurally and functionally similar to other cloned Fur proteins from other bacterial species.

Amino Acid Sequence↗

Baculovirus diversity and molecular biology.

With the identification and characterization of a number of structural and nonstructural protein genes, advances have been made in our understanding of baculovirus structure, regulation of gene expression, and replication. Since less than 30% of the AcMNPV genome has been sequenced and characterized, the continued identification and assignment of function to baculovirus genes is perhaps the most crucial of enterprises now facing baculovirologists and is critical to the development of our understanding of the baculovirus genome and its replication. The size and diversity of baculovirus genomes appears to be strongly influenced by mobile DNA from the insect host. Also, transposon-mediated mutations of baculoviruses provide examples of functional inactivation of viral genes (FP phenotype mutations) and transcriptional activation (TE-D insertion). Another role transposable elements may play is the introduction of insect promoters and enhancers to the baculovirus genome. Since early baculovirus genes are likely transcribed in a way similar to normal insect genes, transposons that insert strong constitutive promoters or cellular enhancers near early baculovirus genes may cause mutations that are subsequently selected for. If this does occur, baculovirus early gene promoters may exhibit a great deal of variability in sequence and may resemble host promoters. Given the overall similarity between the genomes of OpMNPV and AcMNPV and the apparent absence of a region, similar to the AcMNPV HindIII-K/EcoR1-S in OpMNPV, it is intriguing to speculate that this region which contains two ORFs and the hr5 enhancer, may have been inserted into the AcMNPV genome by transposition, possibly delivering several helpful genes (35k and 94k) and a powerful enhancer. The highly repeated enhancer may have been subsequently amplified by recombination. In such a model, the acquisition of general or species-specific enhancers might influence both virulence and host range. Acquisition of general enhancers could increase the level of early gene expression, thus accelerating the cellular infection cycle and making the virus more virulent. Similarly, the acquisition of species-specific enhancers might affect host range by accelerating the infection cycle, but only in a specific host or cell type. One might therefore postulate that diversity in baculoviruses may reflect not only different selection pressures but also the diversity of mobile DNA within host insect species. Although our understanding of baculovirus diversity and molecular biology is rapidly advancing, many of the fundamental characteristics that define the unique nature of baculoviruses remain poorly understood. One fundamental feature of baculoviruses is the production of the two virion phenotypes, PDV and BV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Alpha 1-antitrypsin deficiency caused by the alpha 1-antitrypsin Nullmattawa gene. An insertion mutation rendering the alpha 1-antitrypsin gene incapable of producing alpha 1-antitrypsin.

alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder associated with reduced serum alpha 1AT levels and the development of pulmonary emphysema. An alpha 1AT gene is defined as "Null" when no alpha 1AT in serum is attributed to that alpha 1AT gene. Although all alpha 1AT Null genes have identical phenotypic consequences (i.e. no detectable alpha 1AT in the serum), different genotypic mechanisms can cause the Null state. This study defines the molecular basis for the alpha 1AT gene Nullmattawa, identified and cloned from genomic DNA of an individual with the Null-Null phenotype and emphysema resulting from the heterozygous inheritance of the Nullmattawa and Nullbellingham genes. Sequencing of exons Ic-V and all exon-intron junctions of the Nullmattawa gene demonstrated it was identical to the common normal M1(Val213) alpha 1AT gene except for the insertion of a single nucleotide within the coding region of exon V, causing a 3' frameshift with generation of a premature stop signal. Family analysis using oligonucleotide probes specific for the Nullmattawa sequence demonstrated the gene was inherited in an autosomal fashion. Examination of blood monocytes demonstrated that a normal-sized, 1.8-kb alpha 1AT mRNA transcript is associated with the Nullmattawa gene and in vitro translation of mRNA with the Nullmattawa mutation showed it translated at a normal rate but produced a truncated alpha 1AT protein. Additionally, retroviral transfer of the alpha 1AT Nullmattawa cDNA to murine fibroblasts demonstrated no detectable intracellular or secreted alpha 1AT, despite the presence of alpha 1AT Nullmattawa mRNA transcripts. These findings are consistent with the concept that the molecular pathophysiology of Nullmattawa is likely manifested at a posttranslational level. The identification of the Nullmattawa gene supports the concept that Null alpha 1AT alleles represent a heterogenous group in which very different mechanisms cause the identical phenotypic state.

Adult↗

Identification of an unknown promoter, OUTIIp, within the IS10R element.

A novel promoter in IS10R (OUTIIp) has been found in one of its ends in an inverted position relative to promoter pOUT. OUTIIp shows characteristics similar to those of rpoS-dependent promoters such as a gearbox expression pattern. It is under catabolite repression and positively regulated by ppGpp or conditioned media. This opens new challenges in IS10R transposition.

Bacterial Proteins↗

Identification and activity of two insertion sequence elements in Rhodococcus sp. strain IGTS8.

Two putative insertion sequence (IS) elements, IS1166 and IS1295, were identified on a plasmid present in Rhodococcus sp. IGTS8. Four copies of IS1166 were present in strain IGTS8: one copy on each of two separate plasmids and two copies on a third plasmid or in the chromosome. Of eight rhodococci tested, only R. zopfii contained a copy of an IS1166-like element. Two mutants of strain IGTS8 were isolated in which an additional copy of IS1166 was present, suggesting that at least one copy of this element may be able to transpose. IS1166 and IS1295 are new members of a family of IS elements which includes IS6120 from Mycobacterium smegmatis, IST2 from Thiobacillus ferrooxidans, IS256 from Staphylococcus aureus and ISRm3 from Rhizobium meliloti. The 24-25-bp inverted repeats of these six elements are highly similar and, with the exception of IS1295, their transposases exhibit moderate identities. In particular, the predicted amino-acid sequence of the transposase from IS1166 is 86% identical to that from the known transposable element IS6120.

Amino Acid Sequence↗

Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon.

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.

Bacterial Proteins↗