F1 Genome Research Articles

The puzzle of two tandem acyl-CoA ligases of Pseudomonas putida F1.

Pseudomonas bacteria demonstrate a great deal of metabolic diversity and consequently colonize a wide range of ecological niches. A characteristic of these bacteria is a pair of genes in tandem annotated as acyl-CoA ligases involved in fatty acid degradation. The Pseudomonas putida F1 genome is annotated as having at least nine genes encoding acyl-CoA ligases which are scattered around the chromosome excepting the tandem pair. Since similar tandem pairs are found in other pseudomonads, we have constructed and characterized deletion mutants of the tandem ligases. We report that the encoded proteins are authentic acyl-CoA ligases involved in fatty acid degradation.

A cryptic long-chain 3-ketoacyl-ACP synthase in the Pseudomonas putida F1 unsaturated fatty acid synthesis pathway

The Pseudomonas putida F1 genome contains five genes annotated as encoding 3-ketoacyl-acyl carrier protein (ACP) synthases. Four are annotated as encoding FabF (3-ketoacyl-ACP synthase II) proteins, and the fifth is annotated as encoding a FabB (3-ketoacyl-ACP synthase I) protein. Expression of one of the FabF proteins, FabF2, is cryptic in the native host and becomes physiologically important only when the repressor controlling fabF2 transcription is inactivated. When derepressed, FabF2 can functionally replace FabB, and when expressed from a foreign promoter, had weak FabF activity. Complementation of Escherichia coli fabB and fabF mutant strains with high expression showed that P. putida fabF1 restored E. coli fabF function, whereas fabB restored E. coli fabB function and fabF2 restored the functions of both E. coli fabF and fabB. The P. putida ΔfabF1 deletion strain was almost entirely defective in synthesis of cis-vaccenic acid, whereas the ΔfabB strain is an unsaturated fatty acid (UFA) auxotroph that accumulated high levels of spontaneous suppressors in the absence of UFA supplementation. This was due to increased expression of fabF2 that bypasses loss of fabB because of the inactivation of the regulator, Pput_2425, encoded in the same operon as fabF2. Spontaneous suppressor accumulation was decreased by high levels of UFA supplementation, whereas competition by the P. putida β-oxidation pathway gave increased accumulation. The ΔfabB ΔfabF2 strain is a stable UFA auxotroph indicating that suppressor accumulation requires FabF2 function. However, at low concentrations of UFA supplementation, the ΔfabF2 ΔPput_2425 double-mutant strain still accumulated suppressors at low UFA concentrations.

Isolation and characterization of a bacterial strain that degrades cis-dichloroethenein the absence of aromatic inducers.

Bacteria capable of degrading cis-dichloroethene (cDCE) were screened from cDCE-contaminated soil, and YKD221, a bacterial strain that exhibited a higher growth on minimal salt agar plates in the presence of cDCE than in the absence of cDCE, were isolated. Phylogenetic studies of the 16S rRNA as well as gyrB, rpoD, and recA in YKD221 indicated that this strain is closely related to the type strains of Pseudomonas plecoglossicida, monteilii, and putida. An average nucleotide identity analysis indicated that YKD221 is most closely related to P. putida strains, including the type strain, which suggests that YKD221 belongs to P. putida. Although the genome of YKD221 was very similar to that of P. putida F1, a toluene-degrading strain, the YKD221 genome has 15 single-nucleotide polymorphisms and 4 insertions compared with the F1 genome. YKD221 caused the release of sufficient chloride ions from cDCE to suggest that the strain is able to completely dechlorinate and degrade cDCE. YKD221 also degraded trichloroethene but was unable to degrade trans-dichloroethene and tetrachloroethene. The degradation activity of YKD221 was elevated after growth on toluene. Inactivation of todC1, which encodes for a large subunit of the catalytic terminal component in toluene dioxygenase, resulted in a complete loss of growth on toluene and cDCE degradation activity. This is the first evidence of the involvement of todC1C2BA-coded toluene dioxygenase in cDCE degradation. YKD221 did not appear to grow on cDCE in a minimal salt liquid medium. However, YKD221 did exhibit an enhanced increase in cell concentration and volume of cells during growth on minimal salt agar plates with cDCE when first grown in LB medium. This behavior appears to have led us to misinterpret our initial results on YKD221 as an indication of improved growth in the presence of cDCE.

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506bp in length with an average G+C content of 61.9%. The genome contains 94 putative open reading frames (ORFs) and 74.5% of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5% (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

F1 and Tbilisi Are Closely Related Brucellaphages Exhibiting Some Distinct Nucleotide Variations Which Determine the Host Specificity

We report on the 41,143-bp genome of brucellaphage F1, a podovirus that infects several Brucella species. The F1 genome is almost identical to the genome of brucellaphage Tb. However, some structural proteins of the phages exhibit extensive polymorphisms and might be responsible for their different host ranges.

Integrating Grant‐funded Research into the Undergraduate Biology Curriculum Using IMG‐ACT

It has become clear in current scientific pedagogy that the emersion of students in the scientific process in terms of designing, implementing, and analyzing experiments is imperative for their education; as such, it has been our goal to model this active learning process in the classroom and laboratory in the context of a genuine scientific question. Toward this objective, the National Science Foundation funded a collaborative research grant between a primarily undergraduate institution and a research-intensive institution to study the chemotactic responses of the bacterium Pseudomonas putida F1. As part of the project, a new Bioinformatics course was developed in which undergraduates annotate relevant regions of the P. putida F1 genome using Integrated Microbial Genomes Annotation Collaboration Toolkit, a bioinformatics interface specifically developed for undergraduate programs by the Department of Energy Joint Genome Institute. Based on annotations of putative chemotaxis genes in P. putida F1 and comparative genomics studies, undergraduate students from both institutions developed functional genomics research projects that evolved from the annotations. The purpose of this study is to describe the nature of the NSF grant, the development of the Bioinformatics lecture and wet laboratory course, and how undergraduate student involvement in the project that was initiated in the classroom has served as a springboard for independent undergraduate research projects.

Identification of a New HIV Type 1 BF Intersubtype Circulating Recombinant Form (CRF44_BF) in Chile

HIV-1 BF intersubtype recombinants are frequent in Argentina, Uruguay, and Brazil, where among a high diversity of BF unique recombinant forms (URFs), eight circulating recombinant forms (CRFs) have been characterized. Here, we describe a new one, designated CRF44_BF, identified in HIV-1 samples from Chile. In a previous report, where partial pol sequences of 136 HIV-1 infections of Chilean subjects were analyzed, a phylogenetic cluster of HIV-1 recombinant BF viruses from 10 individuals, with coincident intersubtype recombination points, was detected. One virus of this cluster had been characterized along its near full-length genome. A second one, from an epidemiologically unlinked HIV-1-infected subject, is described here. Both genomes share identical mosaic structures, consisting of a predominantly subtype F1 genome with three fragments of subtype B. Coincident breakpoints and phylogenetic clustering of the newly identified CRF44_BF with CRF12_BF, CRF17_BF, and CRF38_BF support a common origin of different CRF_BFs identified in Argentina, Uruguay, and Chile.

An extension of the Minute Virus of Mice tissue tropism

Well-defined tissue tropism makes Autonomous Parvoviruses a valuable model for studies of virus–cell interactions and gene therapy research. We developed a new Minute Virus of Mice variant, different from the known prototype (MVMp) and immunosuppressive (MVMi) strains. The new virus variant, designated F1, was isolated from the culture of semi-permissive Fisher Rat Fibroblasts, F111, infected with MVMp. The F1 genome carried point mutations in regions known to determine the mutually restricted host ranges of MVMp and MVMi. In F111 cells, F1 cytotoxicity, gene expression and multiplication were significantly higher compared to MVMp. Conversely the wild-type virus propagated in MVMp-permissive cells more efficiently than the F1. Reversion of the F1-specific mutations to wild-type MVMp sequence, following reverse-passaging of the mutant virus in MVMp-permissive cells, confirmed a specific adaptation of the F1 virus to F111 cells. Considerable divergence in tissue specificities between the wild-type and mutant viruses was demonstrated in vivo.

Cytogenetics of a new trispecies hybrid in cotton: [(Gossypium hirsutum L. × G. thurberi Tod.)2 × G. longicalyx Hutch. & Lee

AbstractA three‐species hybrid named HTL including Gossypium hirsutum L. [2n = 4 x = 52, (AD)1 genome] was created using the pseudophyletic introgression method with G. longicalyx Hutch. & Lee (2n = 2x = 26, F1 genome) as donor parent and G. thurberi Tod. (2n = 2x = 26, D1 genome) as bridge species. The new hybrid was totally self‐sterile and its interspecific status was confirmed using simple sequence repeat markers and cytogenetic analysis. Cytogenetic studies showed that its chromosome configuration was 2n = 52 = 14.13 I + 15.10 II + 1.03 III + 0.9 IV + 0.03 V + 0.13 VI (where I, II, III, IV, V and VI are univalents, bivalents, trivalents, tetravalents, pentavalents and hexavalents, respectively). Prospects for successfully exploiting the HTL hybrid in breeding programmes are discussed.

Appropriate expression of filamentous phage f1 DNA replication genes II and X requires RNase E-dependent processing and separate mRNAs.

The products of in-frame overlapping genes II and X carried by the filamentous phage f1 genome are proteins with required but opposing functions in phage DNA replication. Their normal relative levels are important for continuous production of phage DNA without killing infected Escherichia coli hosts. Here we identify several factors responsible for determining the relative levels of pII and pX and that, if perturbed, alter the normal distribution of the phage DNA species in infected hosts. Translation of the two proteins is essentially relegated to separate mRNAs. The mRNAs encoding genes II and X are also differentially sensitive to cleavage dependent on rne, the gene encoding the only E. coli endo-RNase known to have a global role in mRNA stability. Whereas pII levels are limited at the level of mRNA stability, normal pX levels require transcription in sufficient amounts from the promoter for the smaller mRNA encoding only pX.

Nuclear DNA content in F1 hybrids of maize

The nuclear DNA content was determined in two separate experiments for 19 maize inbred lines and 26 maize F1 hybrids. Ten inbred lines were initially screened in the first experiment. An 11 per cent difference in nuclear DNA amount was observed between the two lines with the lowest amounts of DNA and two lines with the largest genome sizes. All possible hybrid combinations were made among these four lines. In all cases, the genome sizes of the F1 hybrids were not significantly different from their expected parental means. In several cases, however, F1 plants of specific crosses were not uniform in genome size. In these crosses, genome sizes observed ranged from the genome size of the low parental genome to the largest parental genome. These results indicate an instability in F1 genome sizes in certain maize crosses. In order to corroborate this hypothesis, a second experiment was performed. To remove any biases with respect to genome size, 14 F1 maize hybrids were selected solely on the basis of their heterotic response with no regard for their genome sizes or the genome sizes of their parental inbred lines. The nuclear DNA content of the nine parental lines and 14 hybrids was determined. In most of the crosses, the nuclear DNA content of the F1 hybrids was not significantly different from their respective parental means. However, in five parental combinations, the mean nuclear DNA content of the F, hybrids was significantly higher than their respective parental means. The combined results of this study support the hypothesis of instability in nuclear DNA content in F1 hybrids of maize. This instability appears not to be universal in all maize hybrids but is restricted to specific parental combinations.

9] pEMBL: A new family of single-stranded plasmids for sequencing DNA

This chapter describes a series of plasmids, the “pEMBL family,” that have the property of being encapsidated as single-stranded deoxyribonucleic acid (DNA) upon superinfection with phage F1. The plasmids can be used for DNA sequencing and for all the other uses that require single-stranded DNA, but, in the absence of F1 helper phage, they replicate as any other doubles-stranded plasmid. This is very useful for the preparation of a large quantity of plasmid and for the stability of longer inserts. The class of M13 derivative vectors, largely used for DNA sequencing with the dideoxy method, has, in fact, the limitation that large inserts tend to be unstable. The chapter discusses the principle of the method and characteristics of pEMBL vectors along with the construction of pEMBL plasmids. The chapter discusses the property of a short segment of F1 genome containing all the cis-acting elements for DNA replication and morphogenesis. To have a series of polylinkers containing a large number of unique sites for restriction endonucleases, in different reciprocal orientation is very useful, because it allows the insertion of DNA fragments in a preselected orientation or in a preselected frame inside the sequence encoding the a-fragment of th/3-galactosidase. The DNA, which is prepared through the procedures described in the chapter, is suitable for DNA sequencing experiments with the dideoxy method.

The bacteriophage f1 morphogenetic signal and the gene V protein/phage single-stranded DNA complex

The region of the bacteriophage f1 genome near the gene IV/intergenic region (IG) junction previously was shown to contain a sequence necessary for efficient packaging of single-stranded (SS) DNA into phage particles (the f1 “morphogenetic signal”) [ G. P. Dotto, V. Enea, and N. D. Zinder (1981) Virology 114, 463–473]. The DNA content of f1 phage/pBR322 chimeric plasmid-transformed, phage-infected bacteria has been investigated. The chimeric plasmids, constructed by Dotto et al. (1981) , contained the f1 origins of DNA replication and, in some cases, also contained the “morphogenetic signal.” Chimeric plasmid SS DNA was detected in phage-infected bacteria harboring any one of these chimeric plasmids, and the majority of this SS DNA was found complexed to the phage gene V protein. Therefore, the “morphogenetic signal” is not required for the formation of the gene V protein/f1 SS DNA complex but instead must function at a later stage of filamentous phage morphogenesis.

MRNA processing inEscherichia coli: an activity encoded by the host processes bacteriophage f1 mRNAs

To examine the regions of the male-specific filamentous bacteriophage f1 genome that include signals for mRNA processing, the 5' endpoints of the major in vivo phage mRNAs have been located in the f1 DNA sequence by S1 nuclease mapping. The 5' ends of the purified mRNAs and additional phage-specific RNAs transiently visible early after infection occur in clusters of T-rich residues within genes that code for three phage proteins. When a 270-nucleotide region encompassing the 5' endpoints of three processed RNAs is transcribed as part of the bacteriophage lambda N mRNA in uninfected female cells, RNA 5' ends identical to ends of the three f1 RNAs are generated from the lambda-f1 precursor. This finding indicates that the mRNA processing activity is encoded by the bacterial host, and that its recognition sites are present in the local regions near the 5' ends which result from RNA cleavage. Several characteristics of f1 mRNA processing events have implications for the differential regulation of adjacent phage genes constrained in the same transcription unit, and may be representative of similar processing events occurring in the bacterial cell.

Co-evolution of a filamentous bacteriophage and its defective interfering particles

Serial passage of bacteriophage f1 at high multiplicities of infection results in the appearance of defective deletion mutants (miniphage) that harbor a tandem reiteration of regions of the f1 genome near the origin of DNA replication. These miniphage interfere with the growth of wild-type f1, and cause a sharp decrease of the viable phage titer. Upon further passage, however, the titer increases again. Viable phage variants (maxiphage) appear which harbor the same tandem reiteration of DNA as the miniphage. The maxiphage are more resistant than the wild type to interference by the miniphage. In the absence of miniphage the maxiphage grow at the same rate as the wild type. The structure of the DNA reiteration gradually changes during further passage. Miniphage and maxiphage follow, in parallel, a similar course of changes in the pattern of reiteration. In miniphage the reiterations change while the deletions are conserved. Serial passage of maxiphage quickly yields miniphage, which harbor a reiteration identical to that of the parental maxiphage. Both reiteration and deletion are relevant to the mechanism of interference by miniphage. Thus serial passage of the filamentous phage affords an experimental system to study evolution of a DNA genome in test tubes. Possible mechanisms of the interference by miniphage are discussed.

One and two codon insertion mutants of bacteriophage f1

Simple methods for introducing one or two extra codons of genetic information into the f1 genome in vitro have been devised. The methods use various combinations of enzymes to insert three or six base-pairs into the RF1 DNA of the bacteriophage. Since such insertions do not cause frameshifts in coding regions, a number of these mutants are viable. Several such mutants were mapped and characterized. The methods described and variations of them can be applied to other circular DNA genomes.

Restructuring the bacteriophage f1 genome: Expression of gene VIII in the intergenic space

Using in vitro recombinant DNA techniques, we have constructed a derivative of bacteriophage f1 which contains an amber mutation in the major coat protein gene (gene VIII) plus an additional wild-type copy of this gene in the intergenic space near the origin of phage DNA replication. The gene is functional in this new location and is expressed at the same level relative to available transcriptional template DNA (RF) as the corresponding gene in wild-type f1. The implication of this finding in terms of current models of gene expression in the filamentous coliphages is discussed. Intertypic mixing of coat protein monomers derived from the two genes occurs when this phage is grown in an amber suppressor host. The effect of this DNA insertion on replication of the f1 genome is discussed. The concept of restructuring the f1 genome as a means of studying its regulation and expression is also discussed.

Insertion mutant of bacteriophage f1 sensitive to EcoRI.

The nucleotide sequence A-A-T-T was inserted into the intergenic region of the f1 genome at a site cleaved by Hae III (cleavage sequence (G-G-C-C). The resultant viable phage mutant (R199) contains a single site sensitive to the restriction endonuclease EcoRI (cleavage sequence G-A-A-T-T-C). This phage is sensitive to EcoRI restriction and modification in vivo and in vitro. Its potential for use as a cloning vector has been tested by construction in vitro of an f1/pBR322 chimeric phage. The four bases inserted into wild-type f1 to generate the R199 mutant came from a small restriction fragment obtained by digesting plasmid pBR322 with EcoRI and HindIII. The use of this linker prepared from a biological substrate is an example of a technique for constructing restriction enzyme sites in vitro. It is presented as an alternative to the use of synthetic linkers and should be generally applicable.

Cleavage map of bacteriophage f1: Location of the Escherichia coli B-specific modification sites

Replicative form DNA of bacteriophage f1 was cleaved into specific fragments by two endonucleases isolated from Hemophilus aegyptius and an endonuclease isolated from H. influenzae. The fragments were ordered so as to construct a circular map of the phage f1 genome by: (1) digesting the isolated restriction fragments with a second restriction enzyme; and (2) testing in a transfection system for the ability of the fragments to rescue amber mutations contained on single-stranded viral DNA that was hybridized to a particular fragment. The genome of bacteriophage f1 contains two SB sites, genetic sites which confer upon a DNA molecule susceptibility to the restriction-modification system of Escherichia coli B. Each of these sites was located on a specific restriction fragment by transfection experiments. In vitro modification of replicative form DNA of f1 and its SB mutants by endonuclease R · EcoB and the subsequent cleavage of the DNA by the Hemophilus endonucleases showed that the B-specific methylation occurs within at least 200 nucleotide pairs of the SB site.