Putative Transposase Research Articles

Genomic characterization of antibiotic-resistant Staphylococcus epidermidis with observed shifts in optimal temperature.

Antibiotic resistance genes (ARGs) in the environment pose significant public health concerns and are influenced by conditions like temperature changes. We previously observed that resistance evolution to gentamicin and colistin affects optimal growth temperatures in Staphylococcus epidermidis isolates. Despite significant phenotype observations, the genetic basis remains unclear. We aim to identify the genetic changes linked to antibiotic resistance evolution that alter optimal growth temperature. Using whole-genome sequencing, we sequenced the genomes of gentamicin-resistant (GEN-1, GEN-2) and colistin-resistant (COL-4, COL-6) S. epidermidis isolates. Variant analysis with the BV-BRC bioinformatics tool identified genes involved in antibiotic resistance and temperature response. We found 12 genetic variants, including two unique to GEN-2 and one in COL-4. One shared mutation was observed in GEN-1 and GEN-2, and another in COL-4 and COL-6. Five mutations were shared among all isolates related to mobile gene elements, including a transposase IS4 family, two putative transposases, and two transposase-like insertion elements. Our findings indicate that the same genes involved in gentamicin and colistin resistance, especially those related to mobile genetic elements, may also play a crucial role in temperature response.

AcademH, a lineage of Academ DNA transposons encoding helicase found in animals and fungi

BackgroundDNA transposons are ubiquitous components of eukaryotic genomes. Academ superfamily of DNA transposons is one of the least characterized DNA transposon superfamilies in eukaryotes. DNA transposons belonging to the Academ superfamily have been reported from various animals, one red algal species Chondrus crispus, and one fungal species Puccinia graminis. Six Academ families from P. graminis encode a helicase in addition to putative transposase, while some other families encode a single protein which contains a putative transposase and an XPG nuclease.ResultsSystematic searches on Repbase and BLAST searches against publicly available genome sequences revealed that several species of fungi and animals contain multiple Academ transposon families encoding a helicase. These AcademH families generate 9 or 10-bp target site duplications (TSDs) while Academ families lacking helicase generate 3 or 4-bp TSDs. Phylogenetic analysis clearly shows two lineages inside of Academ, designated here as AcademH and AcademX for encoding helicase or XPG nuclease, respectively. One sublineage of AcademH in animals encodes plant homeodomain (PHD) finger in its transposase, and its remnants are found in several fish genomes.ConclusionsThe AcademH lineage of TEs is widely distributed in animals and fungi, and originated early in the evolution of Academ DNA transposons. This analysis highlights the structural diversity in one less studied superfamily of eukaryotic DNA transposons.

Complex Evolutionary History of Mboumar, a Mariner Element Widely Represented in Ant Genomes

Mboumar-9 is an active mariner-transposable element previously isolated in the ant Messor bouvieri. In this work, a mariner-like element, Mboumar, isolated from 22 species of ants, is analyzed. These species belong to nine different subfamilies, including Leptanillinae, the most primitive ant subfamily, and Myrmicinae and Formicidae, the most derived ones. Consequently, Mboumar-like elements seem to be well-represented in ant genomes. The phylogenetic tree drawn for mariner elements is highly inconsistent with the phylogeny of host ants, with almost identical elements found in clearly distant species and, on the contrary, more variable elements in closely related species. The inconsistency between the two phylogenetic trees indicates that these transposable elements have evolved independently from the speciation events of the ants that host them. Besides, we found closer genetic relationships among elements than among their host ants. We also found potential coding copies with an uninterrupted open reading frame of 345 aa in 11 species. The putative transposase codified by them showed a high sequence identity with the active Mboumar-9 transposase. The results of selection tests suggest the intervention of purifying selection in the evolution of these elements. Overall, our study suggests a complex evolutionary history of the Mboumar-like mariner in ants, with important participation of horizontal transfer events. We also suggest that the evolutionary dynamics of Mboumar-like elements can be influenced by the genetic system of their host ants, which are eusocial insects with a haplodiploid genetic system.

Transposition of IS4 Family Insertion Sequences ISTeha3, ISTeha4, and ISTeha5 into the arc Operon Disrupts Arginine Deiminase System in Tetragenococcus halophilus.

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is often used as a starter culture in the manufacturing of soy sauce. T. halophilus possesses an arginine deiminase system, which is responsible for the accumulation of citrulline, the main precursor of the potential carcinogen ethyl carbamate. In this study, we generated five derivatives lacking arginine deiminase activity from T. halophilus NBRC 12172 by UV irradiation. Using these derivatives as a fermentation starter prevented arginine deimination in soy sauce. DNA sequence analysis of the derivatives revealed that novel IS4 family insertion sequences, designated ISTeha3, ISTeha4, and ISTeha5, were transposed into the region around the arginine deiminase (arc) operon in the mutants. These insertion sequences contain a single open reading frame encoding a putative transposase and 13- to 15-bp inverted repeats at both termini, which are adjacent to 7- to 9-bp duplications of the target sequence. Investigation of wild strains isolated from soy sauce mash incapable of arginine deimination also indicated that insertion sequences are involved in the disruption of the arginine deiminase system in T. halophilusIMPORTANCE Insertion sequences play important roles in bacterial evolution and are frequently utilized in mutagenesis systems. However, the intrinsic insertion sequences of tetragenococci are not well characterized. Here, we identified three active insertion sequences of T. halophilus by transposition into the region around the arc operon. This report provides an example of insertion sequence-mediated generation and evolution of T. halophilus and primary information about their characteristics.

Potential use of a group II alphabaculovirus isolated from Mamestra brassicae as a broad-spectrum biological pesticide

ABSTRACTA novel native isolate of Mamestra brassicae nucleopolyhedrovirus, designated MabrNPV-CHb1, was identified in this study. The complete nucleotide sequence of MabrNPV-CHb1 was determined. The MabrNPV-CHb1 genome consists of 154,451 bp, with a G + C content of 40.05%. The genomic sequence and comparative analysis indicated that MabrNPV-CHb1 shares high nucleotide identity with MabrNPV-K1, HearMNPV, MacoNPV-B and MacoNPV-A. The major difference between MabrNPV-CHb1 and MabrNPV-K1 is the absence of MabrNPV-CHb1 ORF134 in MabrNPV-K1. The MabrNPV-CHb1 ORF134 is a gene encoding a putative transposase, and phylogenetic analysis suggests that it is derived through horizontal gene transfer (HGT) from ascoviruses. MabrNPV-CHb1 exhibits broad-spectrum activity against lepidopteran agricultural pests, including insects in the families Noctuidae and Plutellidae. A high yield of MabrNPV-CHb1 occlusion bodies was obtained by propagation in alternative hosts, demonstrating its potential for pesticide application. Its broad host range, high yields in alternative hosts and clear genetic background could extend its potential applications as a broad-spectrum insecticide in the future.

Genetic analysis of a PER-2-producing Shewanella sp. strain harbouring a variety of mobile genetic elements and antibiotic resistance determinants.

The objective of this study was to investigate the molecular mechanisms explaining the multidrug-resistant (MDR) phenotype found in a novel clinical Shewanella sp. strain (Shew256) recovered from a diabetic patient. Whole-genome shotgun sequencing was performed using Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPAdes. RAST Server was used to predict the open-reading frames and the predictions were confirmed using BLAST. Further genomic analysis was carried out using average nucleotide identity (ANI), ACT (Artemis), OrthoMCL, ARG-ANNOT, ISfinder, PHAST, tRNAscan-SE, plasmidSPAdes, PlasmidFinder and MAUVE. PCR and plasmid extraction were also performed. Genomic analysis revealed a total of 456 predicted genes unique to Shew256 compared with other Shewanella genomes. Moreover, the presence of different resistance genes, including blaPER-2, was found. A complex class 1 integron containing the ISCR1 gene, disrupted by two putative transposase genes, was identified. Furthermore, other resistance genes, a transposon containing aph(3'), insertion sequences, phages and non-coding RNAs were also found. In conclusion, evidence of acquisition of resistance genes and mobile elements that could explain the MDR phenotype were observed. This Shewanella sp. represents a prime example of how antibiotic resistance determinants can be acquired by uncommon pathogens.

Gene Overexpression in Streptomyces hygroscopicus Associated with DNA Amplification.

The genetics of the Streptomyces hygroscopicus strain 10-22 is of interest due to the ability of this strain to produce antifungal compounds. Strain T110 was obtained through insertional mutagenesis of strain 10-22 and was found to have undergone DNA amplification, as determined by both conventional and pulsed-field gel electrophoresis (PFGE). pIJ702, the vector used for insertional mutagenesis, was shown to have integrated into and co-amplified with the chromosomal DNA sequence of T110, as pIJ702 hybridized predominantly with two of the three amplified BamHI fragments. The amplified DNA sequence in T110 is 10.8kb in length and consists of 5.18kb of Streptomyces chromosomal DNA and the entire 5.62kb pIJ702 sequence. Sequence analysis of the 5.18kb chromosomal sequence revealed two open reading frames, one encoding a putative IS5 family transposase and the other encoding a putative dihydroxy-acid dehydratase. Real-time PCR analysis showed that expression of the putative dehydratase gene in T110 is about 50 times greater than in the wild-type strain, consistent with the high level of amplification of this DNA region, and therefore this system has the potential for producing economically or clinically important molecules.

Variants of astA gene among extra-intestinal Escherichia coli of human and avian origin.

Many Escherichia coli strains harbour astA, which is the gene encoding the enteroaggregative E. coli heat-stable enterotoxin (EAST1). This gene is embedded in a putative transposase (ORF1) and presents polymorphism in diarrheagenic strains. Although astA and orf1 are detected in extraintestinal strains, little is known about polymorphism and differential gene transcription in this pathotype. In the present work, extraintestinal E. coli from humans (ExPEC - Extraintestinal Pathogenic E. coli) and poultry (APEC - Avian Pathogenic E. coli) were assayed to verify the presence of astA/orf1 and possible polymorphisms in these genes. Three astA/orf1 patterns were detected via Sanger sequencing. Pattern 1 was novel and represented an astA pseudogene. Pattern 2 and pattern 3 presented distinct amino acids within the reading frame encoding astA and were identical to the sequences found in EAEC 17-2 and EAEC 042, respectively. Regarding the frame encoding ORF1, all mutations detected in the three patterns were neutral. The transcripts of astA/orf1 in vitro were underregulated in strains possessing the pattern 1 sequence. The results demonstrate that the same astA sequences may be detected in diarrheagenic and extra-intestinal E. coli. However, extraintestinal isolates may also present an astA pseudogene that has not been reported in diarrheagenic E. coli.

PARP-1 is required for retrieval of cocaine-associated memory by binding to the promoter of a novel gene encoding a putative transposase inhibitor.

Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and short hairpin RNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin immunoprecipitation sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified among PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment markedly increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse.

Emergence of mixed infection of Beijing/Non-Beijing strains among multi-drug resistant Mycobacterium tuberculosis in Pakistan.

Tuberculosis (TB) remains as one of the deadliest diseases after HIV globally with 95 % of deaths confined to low-and-middle income countries. Pakistan is fifth among the 22 high-burden TB countries with the incidence rate of 230/100,000 persons, however, studies related to prevalent Mycobacterium tuberculosis strains and their spread, drug resistance pattern and evolutionary genetics are inadequate. The present study was undertaken to highlight the circulation of M. tuberculosis strains causing drug resistant TB in our community by targeting the molecular marker IS6110 and then characterization of these strains as Beijing and Non-Beijing genotypes. Sputum samples from 102 MDR TB suspects from different cities of Punjab were collected and their record was stored in a database. Sputum samples were evaluated by Ziehl Neelson staining and cultured on Lownstein Jensen medium by Modified Petroff’s method. DST was performed for first-line anti-mycobacterial drugs by indirect proportion method. Mycobacterium tuberculosis isolates were investigated for the presence of IS6110 and further identification as Beijing, Non-Beijing or mixed genotype. Percentage of male and female patients was found to be 58.8 and 41.2 % respectively. DST showed resistance of 93 % of isolates to isoniazid and rifampicin. All of the isolates showed positive results for IS6110 amplification. Based on PCR amplification of Beijing and non-Beijing primer sets 4.9 % of the patients showed infection with pure Beijing isolates, 14.7 % with both Beijing and non-Beijing isolates and 80.3 % with pure non-Beijing isolates. Analysis of IS6110 and Beijing sequences showed the presence of putative transposase conserved domain while non-Beijing sequences were epitomized with RAMP_I_III superfamily domain (CRISPR-associated protein family). TB in Pakistan is predominantly caused by Non-Beijing genotypes, but Beijing strains showed incessant circulation in our community as both single and mixed (co-infecting Non-Beijing and Beijing) strains.

The casposon-encoded Cas1 protein from Aciduliprofundum boonei is a DNA integrase that generates target site duplications.

Many archaea and bacteria have an adaptive immune system known as CRISPR which allows them to recognize and destroy foreign nucleic acid that they have previously encountered. Two CRISPR-associated proteins, Cas1 and Cas2, are required for the acquisition step of adaptation, in which fragments of foreign DNA are incorporated into the host CRISPR locus. Cas1 genes have also been found scattered in several archaeal and bacterial genomes, unassociated with CRISPR loci or other cas proteins. Rather, they are flanked by nearly identical inverted repeats and enclosed within direct repeats, suggesting that these genetic regions might be mobile elements (‘casposons’). To investigate this possibility, we have characterized the in vitro activities of the putative Cas1 transposase (‘casposase’) from Aciduliprofundum boonei. The purified Cas1 casposase can integrate both short oligonucleotides with inverted repeat sequences and a 2.8 kb excised mini-casposon into target DNA. Casposon integration occurs without target specificity and generates 14–15 basepair target site duplications, consistent with those found in casposon host genomes. Thus, Cas1 casposases carry out similar biochemical reactions as the CRISPR Cas1-Cas2 complex but with opposite substrate specificities: casposases integrate specific sequences into random target sites, whereas CRISPR Cas1-Cas2 integrates essentially random sequences into a specific site in the CRISPR locus.

High genetic variation and recombination events in the vicinity of non-autonomous transposable elements from ‘Candidatus Liberibacter asiaticus’

Two miniature inverted-repeat transposable elements (MITEs), MCLas-A and MCLas-B, were recently identified from ‘Candidatus Liberibacter asiaticus’ known to be associated with citrus Huanglongbing (HLB, yellow shoot disease). MCLas-A was suggested as an active MITE because of its mobility. The immediate upstream gene of the two MITEs was predicted to be a putative transposase. The goal of this study is to analyze the sequence variation in the upstream putative transposase of MITEs and explore the possible correlation between sequence variation of transposase gene and MITE activity. PCR and sequence analysis showed that 12 sequence types were found in six major amplicon types from 43 representative ‘Ca. L. asiaticus’ isolates from China, the United States and Brazil. Out of the 12 sequence types, three (T4, T5-2, T6) were reported for the first time. Recombination events were found in the two unique sequence types (T5-2 and T6) which were detected in all Brazilian isolates. Notably, no sequence variation or recombination events were detected in the upstream putative transposase gene of MCLas-A, suggesting the conservation of the transposase gene might be closely related with the MITE activity. Phylogenetic analysis demonstrated two well supported clades including five subclades were identified, clearly reflecting the geographical origins of isolates, especially that of Ruili isolates, São Paulo isolates and a few Florida isolates.

Assessment of Probiotics in Infant Formula and Cereal Based Baby Foods Containing Bifidobacterium lactis Bb12– Update 2014

Assessment of Probiotics in Infant Formula and Cereal Based Baby Foods Containing Bifidobacterium lactis Bb12– Update 2014

Adaptation, ecology, and evolution of the halophilic stromatolite archaeon Halococcus hamelinensis inferred through genome analyses.

Halococcus hamelinensis was the first archaeon isolated from stromatolites. These geomicrobial ecosystems are thought to be some of the earliest known on Earth, yet, despite their evolutionary significance, the role of Archaea in these systems is still not well understood. Detailed here is the genome sequencing and analysis of an archaeon isolated from stromatolites. The genome of H. hamelinensis consisted of 3,133,046 base pairs with an average G+C content of 60.08% and contained 3,150 predicted coding sequences or ORFs, 2,196 (68.67%) of which were protein-coding genes with functional assignments and 954 (29.83%) of which were of unknown function. Codon usage of the H. hamelinensis genome was consistent with a highly acidic proteome, a major adaptive mechanism towards high salinity. Amino acid transport and metabolism, inorganic ion transport and metabolism, energy production and conversion, ribosomal structure, and unknown function COG genes were overrepresented. The genome of H. hamelinensis also revealed characteristics reflecting its survival in its extreme environment, including putative genes/pathways involved in osmoprotection, oxidative stress response, and UV damage repair. Finally, genome analyses indicated the presence of putative transposases as well as positive matches of genes of H. hamelinensis against various genomes of Bacteria, Archaea, and viruses, suggesting the potential for horizontal gene transfer.

Exopolysaccharide production and ropy phenotype are determined by two gene clusters in putative probiotic strain Lactobacillus paraplantarum BGCG11.

Lactobacillus paraplantarum BGCG11, a putative probiotic strain isolated from a soft, white, artisanal cheese, produces a high molecular-weight heteropolysaccharide, exopolysaccharide (EPS)-CG11, responsible for the ropy phenotype and immunomodulatory activity of the strain. In this study, a 26.4-kb region originating from the pCG1 plasmid, previously shown to be responsible for the production of EPS-CG11 and a ropy phenotype, was cloned, sequenced, and functionally characterized. In this region 16 putative open reading frames (ORFs), encoding enzymes for the production of EPS-CG11, were organized in specific loci involved in the biosynthesis of the repeat unit, polymerization, export, regulation, and chain length determination. Interestingly, downstream of the eps gene cluster, a putative transposase gene was identified, followed by an additional rfb gene cluster containing the rfbACBD genes, the ones most probably responsible for dTDP-L-rhamnose biosynthesis. The functional analysis showed that the production of the high-molecular-weight fraction of EPS-CG11 was absent in two knockout mutants, one in the eps and the other in the rfb gene cluster, as confirmed by size exclusion chromatography analysis. Therefore, both eps and rfb genes clusters are prerequisites for the production of high-molecular-weight EPS-CG11 and for the ropy phenotype of strain L. paraplantarum BGCG11.

Adenine methylation may contribute to endosymbiont selection in a clonal aphid population.

BackgroundThe pea aphid Acyrthosiphon pisum has two modes of reproduction: parthenogenetic during the spring and summer and sexual in autumn. This ability to alternate between reproductive modes and the emergence of clonal populations under favorable conditions make this organism an interesting model for genetic and epigenetic studies. The pea aphid hosts different types of endosymbiotic bacteria within bacteriocytes which help the aphids survive and adapt to new environmental conditions and habitats. The obligate endosymbiont Buchnera aphidicola has a drastically reduced and stable genome, whereas facultative endosymbionts such as Regiella insecticola have large and dynamic genomes due to phages, mobile elements and high levels of genetic recombination. In previous work, selection toward cold adaptation resulted in the appearance of parthenogenetic A. pisum individuals characterized by heavier weights and remarkable green pigmentation.ResultsSix adenine-methylated DNA fragments were isolated from genomic DNA (gDNA) extracted from the cold-induced green variant of A. pisum using deoxyadenosine methylase (Dam) by digesting the gDNA with the restriction enzymes DpnI and DpnII, which recognize the methylated and unmethylated GATC sites, respectively. The six resultant fragments did not match any sequence in the A. pisum or Buchnera genomes, implying that they came from facultative endosymbionts. The A1 fragment encoding a putative transposase and the A6 fragment encoding a putative helicase were selected for further comparison between the two A. pisum variants (green and orange) based on Dam analysis followed by PCR amplification. An association between adenine methylation and the two A. pisum variants was demonstrated by higher adenine methylation levels on both genes in the green variant as compared to the orange one.ConclusionTemperature selection may affect the secondary endosymbiont and the sensitive Dam involved in the survival and adaptation of aphids to cold temperatures. There is a high degree of adenine methylation at the GATC sites of the endosymbiont genes at 8°C, an effect that disappears at 22°C. We suggest that endosymbionts can be modified or selected to increase host fitness under unfavorable climatic conditions, and that the phenotype of the newly adapted aphids can be inherited.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-999) contains supplementary material, which is available to authorized users.

First Evidence of Mariner-like Transposons in the Genome of the Marine Microalga Amphora acutiuscula (Bacillariophyta)

Mariner-like elements (MLEs) are transposable elements able to move in the host genomes by a "cut and paste" mechanism. They have been found in numerous organisms. We succeeded in amplifying complete and truncated MLEs in the marine diatom Amphora acutiuscula. Full-length MLEs of 2,100bp delimited by imperfect Terminal Inverted Repeats revealed an intact Open Reading Frame, suggesting that the MLEs could be active. The DNA binding domain of the corresponding putative transposase could have two Helix-Turn-Helix and a Nuclear Location Site motifs, and its catalytic domain includes a particular triad of aspartic acids DD43D not previously reported. The number of copies was estimated to be 38, including approximately 20 full-length elements. Phylogenetic analysis shows that these peculiar MLEs differ from plant and other stramenopile MLEs and that they could constitute a new sub-family of Tc1-mariner elements.

A novel (S)-6-hydroxynicotine oxidase gene from Shinella sp. strain HZN7.

Nicotine is an important environmental toxicant in tobacco waste. Shinella sp. strain HZN7 can metabolize nicotine into nontoxic compounds via variations of the pyridine and pyrrolidine pathways. However, the catabolic mechanism of this variant pathway at the gene or enzyme level is still unknown. In this study, two 6-hydroxynicotine degradation-deficient mutants, N7-M9 and N7-W3, were generated by transposon mutagenesis. The corresponding mutant genes, designated nctB and tnp2, were cloned and analyzed. The nctB gene encodes a novel flavin adenine dinucleotide-containing (S)-6-hydroxynicotine oxidase that converts (S)-6-hydroxynicotine into 6-hydroxy-N-methylmyosmine and then spontaneously hydrolyzes into 6-hydroxypseudooxynicotine. The deletion and complementation of the nctB gene showed that this enzyme is essential for nicotine or (S)-6-hydroxynicotine degradation. Purified NctB could also convert (S)-nicotine into N-methylmyosmine, which spontaneously hydrolyzed into pseudooxynicotine. The kinetic constants of NctB toward (S)-6-hydroxynicotine (Km = 0.019 mM, kcat = 7.3 s(-1)) and nicotine (Km = 2.03 mM, kcat = 0.396 s(-1)) indicated that (S)-6-hydroxynicotine is the preferred substrate in vivo. NctB showed no activities toward the R enantiomer of nicotine or 6-hydroxynicotine. Strain HZN7 could degrade (R)-nicotine into (R)-6-hydroxynicotine without any further degradation. The tnp2 gene from mutant N7-W3 encodes a putative transposase, and its deletion did not abolish the nicotine degradation activity. This study advances the understanding of the microbial diversity of nicotine biodegradation.

Transposable elements belonging to the Tc1-Mariner superfamily are heavily mutated in Colletotrichum graminicola

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen.

Global gene expression analysis of long-term stationary phase effects in E. coli K12 MG1655.

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.