Functional Analysis of the Interdependence between DNA Uptake Sequence and Its Cognate ComP Receptor during Natural Transformation in Neisseria Species

Natural transformation is the widespread biological process by which “competent” bacteria take up free DNA, incorporate it into their genomes, and become genetically altered or “transformed”. To curb often deleterious transformation by foreign DNA, several competent species preferentially take up their own DNA that contains specific DUS (DNA uptake sequence) watermarks. Our recent finding that ComP is the long sought DUS receptor in Neisseria species paves the way for the functional analysis of the DUS-ComP interdependence which is reported here. By abolishing/modulating ComP levels in Neisseria meningitidis, we show that the enhancement of transformation seen in the presence of DUS is entirely dependent on ComP, which also controls transformation in the absence of DUS. While peripheral bases in the DUS were found to be less important, inner bases are essential since single base mutations led to dramatically impaired interaction with ComP and transformation. Strikingly, naturally occurring DUS variants in the genomes of human Neisseria commensals differing from DUS by only one or two bases were found to be similarly impaired for transformation of N. meningitidis. By showing that ComP_sub from the N. subflava commensal specifically binds its cognate DUS variant and mediates DUS-enhanced transformation when expressed in a comP mutant of N. meningitidis, we confirm that a similar mechanism is used by all Neisseria species to promote transformation by their own, or closely related DNA. Together, these findings shed new light on the molecular events involved in the earliest step in natural transformation, and reveal an elegant mechanism for modulating horizontal gene transfer between competent species sharing the same niche.     

A two-host fosmid system for functional screening of (meta)genomic libraries from extreme thermophiles

A new cloning system is described, which allows the construction of large-insert fosmid libraries in Escherichia coli and the transfer of the recombinant libraries to the extreme thermophile Thermus thermophilus via natural transformation. Libraries are established in the thermophilic host by site-specific chromosomal insertion of the recombinant fosmids via single crossover or double crossover recombination at the T. thermophilus pyr locus. Comparative screening of a fosmid library constructed from genomic DNA from the thermophilic spirochaete, Spirochaeta thermophila, for clones expressing thermoactive xylanase activity revealed that 50% of the fosmids that conferred xylanase activity upon the corresponding T. thermophilus transformants did not give rise to xylanase-positive E. coli clones, indicating that significantly more S. thermophila genes are functionally expressed in T. thermophilus than in E. coli. The novel T. thermophilus host/vector system may be of value for the construction and functional screening of recombinant DNA libraries from individual thermophilic or extremely thermophilic organisms as well as from complex metagenomes isolated from thermophilic microbial communities.     

Production and Characterization of Novel Recombinant Adeno-Associated Virus Replicative-Form Genomes: A Eukaryotic Source of DNA for Gene Transfer

Conventional non-viral gene transfer uses bacterial plasmid DNA containing antibiotic resistance genes, cis-acting bacterial sequence elements, and prokaryotic methylation patterns that may adversely affect transgene expression and vector stability in vivo. Here, we describe novel replicative forms of a eukaryotic vector DNA that consist solely of an expression cassette flanked by adeno-associated virus (AAV) inverted terminal repeats. Extensive structural analyses revealed that this AAV-derived vector DNA consists of linear, duplex molecules with covalently closed ends (termed closed-ended, linear duplex, or “CELiD”, DNA). CELiD vectors, produced in Sf9 insect cells, require AAV rep gene expression for amplification. Amounts of CELiD DNA produced from insect cell lines stably transfected with an ITR-flanked transgene exceeded 60 mg per 5×10⁹ Sf9 cells, and 1–15 mg from a comparable number of parental Sf9 cells in which the transgene was introduced via recombinant baculovirus infection. In mice, systemically delivered CELiD DNA resulted in long-term, stable transgene expression in the liver. CELiD vectors represent a novel eukaryotic alternative to bacterial plasmid DNA.     

DNA binding by pilins and their interaction with the inner membrane platform of the DNA transporter in Thermus thermophilus

The natural transformation system of Thermus thermophilus has become a model system for studies of the structure and function of DNA transporter in thermophilic bacteria. The DNA transporter in T. thermophilus is functionally linked to type IV pili (T4P) and the major pilin PilA4 plays an essential role in both systems. However, T4P are dispensable for natural transformation. In addition to pilA4, T. thermophilus has a gene cluster encoding the three additional pilins PilA1–PilA3; deletion of the cluster abolished natural transformation but retained T4P biogenesis. In this study, we investigated the roles of single pilins PilA1, PilA2 and PilA3 in natural transformation by mutant studies. These studies revealed that each of these pilins is essential for natural transformation. Two of the pilins, PilA1 and PilA2, were found to bind dsDNA. PilA1 and PilA3 were detected in the inner membrane (IM) but not in the outer membrane (OM) whereas PilA2 was present in both membranes. All three pilins where absent in pilus fractions. This suggests that the pilins form a short DNA binding pseudopilus anchored in the IM. PilA1 was found to bind to the IM assembly platform of the DNA transporter via PilM and PilO. These data are in line with the hypothesis that a DNA binding pseudopilus is connected via an IM platform to the cytosolic motor ATPase PilF.     

Application of the shsp Gene, Encoding a Small Heat Shock Protein, as a Food-Grade Selection Marker for Lactic Acid Bacteria

Plasmid pSt04 of Streptococcus thermophilus contains a gene encoding a protein with homology to small heat shock proteins (A. Geis, H. A. M. El Demerdash, and K. J. Heller, Plasmid 50:53-69, 2003). Strains cured from the shsp plasmids showed significantly reduced heat and acid resistance and a lower maximal growth temperature. Transformation of the cloned shsp gene into S. thermophilus St11 lacking a plasmid encoding shsp resulted in increased resistance to incubation at 60°C or pH 3.5 and in the ability to grow at 52°C. A food-grade cloning system for S. thermophilus, based on the plasmid-encoded shsp gene as a selection marker, was developed. This approach allowed selection after transfer of native and recombinant shsp plasmids into different S. thermophilus and Lactococcus lactis strains. Using a recombinant plasmid carrying an erythromycin resistance (Em^r) gene in addition to shsp, we demonstrated that both markers are equally efficient in selecting for plasmid-bearing cells. The average transformation rates in S. thermophilus (when we were selecting for heat resistance) were determined to be 2.4 × 10⁴ and 1.0 × 10⁴ CFU/0.5 μg of DNA, with standard deviations of 0.54 × 10⁴ and 0.32 × 10⁴, for shsp and Em^r selection, respectively. When we selected for pH resistance, the average transformation rates were determined to be 2.25 × 10⁴ and 3.8 × 10³ CFU/0.5 μg of DNA, with standard deviations of 0.63 × 10⁴ and 3.48 × 10³, for shsp and Em^r selection, respectively. The applicability of shsp as a selection marker was further demonstrated by constructing S. thermophilus plasmid pHRM1 carrying the shsp gene as a selection marker and the restriction-modification genes of another S. thermophilus plasmid as a functional trait.     

Type IV Pilus Biogenesis,Twitching Motility,and DNA Uptake in Thermus thermophilus: Discrete Roles of Antagonistic ATPases PilF,PilT1, and PilT2

Natural transformation has a large impact on lateral gene flow and has contributed significantly to the ecological diversification and adaptation of bacterial species. Thermus thermophilus HB27 has emerged as the leading model organism for studies of DNA transporters in thermophilic bacteria. Recently, we identified a zinc-binding polymerization nucleoside triphosphatase (NTPase), PilF, which is essential for the transport of DNA through the outer membrane. Here, we present genetic evidence that PilF is also essential for the biogenesis of pili. One of the most challenging questions was whether T. thermophilus has any depolymerization NTPase acting as a counterplayer of PilF. We identified two depolymerization NTPases, PilT1 (TTC1621) and PilT2 (TTC1415), both of which are required for type IV pilus (T4P)-mediated twitching motility and adhesion but dispensable for natural transformation. This suggests that T4P dynamics are not required for natural transformation. The latter finding is consistent with our suggestion that in T. thermophilus, T4P and natural transformation are linked but distinct systems.     

Efficient Agrobacterium-mediated genetic transformation method using hypocotyl explants of radish (Raphanus sativus L.)

To investigate the gene function of radish (Raphanus sativus L.), several attempts have been made to generate genetically transformed radish. However, no efficient and relatively simple method for the genetic transformation of radish has been developed to date. In this study, we established an Agrobacterium-mediated genetic transformation method using the hypocotyl-derived explants of radish cultivar “Pirabikku”. Primarily based on the Brassica transformation procedure, we optimized it for radish transformation. Using this system, the transformation efficiency of radish hypocotyl explants by Agrobacterium tumefaciens strain GV3101 harboring pIG121-Hm was 13.3%. The copy number of transfer DNA integrated into the genome was either one or two in the four independent transgenic plants. Two of the four plants exhibited male sterility and did not produce self-pollinated seeds. Examination of the expression of the β-glucuronidase (GUS) gene in T₁ plants from fertile T₀ plants showed that the GUS genes were inherited. The improvement in the genetic transformation in this study might pave the way for accelerated molecular breeding and genetic analysis of radish.     

Evolutionary Relationships of Primary Prokaryotic Endosymbionts of Whiteflies and Their Hosts

Whiteflies (Hemiptera: Sternorrhyncha: Aleyrodidae) are plant sap-sucking insects that harbor prokaryotic primary endosymbionts (P-endosymbionts) within specialized cells located in their body cavity. Four-kilobase DNA fragments containing 16S-23S ribosomal DNA (rDNA) were amplified from the P-endosymbiont of 24 whiteflies from 22 different species of 2 whitefly subfamilies. In addition, 3-kb DNA fragments containing mitochondrial cytB, nd1, and large-subunit rDNA (LrDNA) were amplified from 17 whitefly species. Comparisons of the P-endosymbiont (16S-23S rDNA) and host (cytB-nd1-LrDNA) phylogenetic trees indicated overall congruence consistent with a single infection of a whitefly ancestor with a bacterium and subsequent cospeciation (cocladogenesis) of the host and the P-endosymbiont. On the basis of both the P-endosymbiont and host trees, the whiteflies could be subdivided into at least five clusters. The major subdivision was between the subfamilies Aleyrodinae and Aleurodicinae. Unlike the P-endosymbionts of may other insects, the P-endosymbionts of whiteflies were related to Pseudomonas and possibly to the P-endosymbionts of psyllids. The lineage consisting of the P-endosymbionts of whiteflies is given the designation “Candidatus Portiera” gen. nov., with a single species, “Candidatus Portiera aleyrodidarum” sp. nov.     

Pilin-Like Proteins in the Extremely Thermophilic Bacterium Thermus thermophilus HB27: Implication in Competence for Natural Transformation and Links to Type IV Pilus Biogenesis

The extreme thermophile Thermus thermophilus HB27 exhibits high frequencies of natural transformation. Although we recently reported identification of the first competence genes in Thermus, the molecular basis of DNA uptake is unknown. A pilus-like structure is assumed to be involved. Twelve genes encoding prepilin-like proteins were identified in three loci in the genome of T. thermophilus. Mutational analyses, described in this paper, revealed that one locus, which contains four genes that encode prepilin-like proteins (pilA1 to pilA4), is essential for natural transformation. Additionally, comZ, a new competence gene with no similarity to known genes, was identified. Analysis of the piliation phenotype revealed wild-type piliation of a pilA1-pilA3Δkat mutant and a comZ mutant, whereas a pilA4 mutant was found to be completely devoid of pilus structures. These findings, together with the significant similarity of PilA4 to prepilins, led to the conclusion that the T. thermophilus pilus structures are type IV pili. Furthermore, the loss of the transformation and piliation phenotype in the pilA4 mutant suggests that type IV pili are implicated in natural transformation of T. thermophilus HB27.     

Secondary (γ-Proteobacteria) Endosymbionts Infect the Primary (β-Proteobacteria) Endosymbionts of Mealybugs Multiple Times and Coevolve with Their Hosts

Mealybugs (Hemiptera, Coccoidea, Pseudococcidae) are plant sap-sucking insects that have within their body cavities specialized cells containing prokaryotic primary endosymbionts (P-endosymbionts). The P-endosymbionts have the unusual property of containing within their cytoplasm prokaryotic secondary endosymbionts (S-endosymbionts) [C. D. von Dohlen, S. Kohler, S. T. Alsop, and W. R. McManus, Nature (London) 412:433-436, 2001]. Four-kilobase fragments containing 16S-23S ribosomal DNA (rDNA) were obtained from the P-endosymbionts of 22 mealybug species and the S-endosymbionts of 12 representative species. Phylogenetic analyses of the P-endosymbionts indicated that they have a monophyletic origin and are members of the β-subdivision of the Proteobacteria; these organisms were subdivided into five different clusters. The S-endosymbionts were members of the γ-subdivision of the Proteobacteria and were grouped into clusters similar to those observed with the P-endosymbionts. The S-endosymbiont clusters were distinct from each other and from other insect-associated bacteria. The similarity of the clusters formed by the P- and S-endosymbionts suggests that the P-endosymbionts of mealybugs were infected multiple times with different precursors of the S-endosymbionts and once the association was established, the P- and S-endosymbionts were transmitted together. The lineage consisting of the P-endosymbionts of mealybugs was given the designation “Candidatus Tremblaya” gen. nov., with a single species, “Candidatus Tremblaya princeps” sp. nov. The results of phylogenetic analyses of mitochondrial DNA fragments encoding cytochrome oxidase subunits I and II from four representative mealybug species were in agreement with the results of 16S-23S rDNA analyses, suggesting that relationships among strains of “Candidatus T. princeps” are useful in inferring the phylogeny of their mealybug hosts.     

The mRNA of Human Cytoplasmic Arginyl-tRNA Synthetase Recruits Prokaryotic Ribosomes Independently

There are two isoforms of cytoplasmic arginyl-tRNA synthetase (hcArgRS) in human cells. The long form is a component of the multiple aminoacyl-tRNA synthetase complex, and the other is an N-terminal truncated form (ΔNhcArgRS), free in the cytoplasm. It has been shown that the two forms of ArgRS arise from alternative translational initiation in a single mRNA. The short form is produced from the initiation at a downstream, in-frame AUG start codon. Interestingly, our data suggest that the alternative translational initiation of hcArgRS mRNA also takes place in Escherichia coli transformants. When the gene encoding full-length hcArgRS was overexpressed in E. coli, two forms of hcArgRS were observed. The N-terminal sequencing experiment identified that the short form was identical to the ΔNhcArgRS in human cytoplasm. By constructing a bicistronic system, our data support that the mRNA encoding the N-terminal extension of hcArgRS has the capacity of independently recruiting E. coli ribosomes. Furthermore, two critical elements for recruiting prokaryotic ribosomes were identified, the “AGGA” core of the Shine-Dalgarno sequence and the “A-rich” sequence located just proximal to the alternative in-frame initiation site. Although the mechanisms of prokaryotic and eukaryotic translational initiation are distinct, they share some common features. The ability of the hcArgRS mRNA to recruit the prokaryotic ribosome may provide clues for shedding light on the mechanism of alternative translational initiation of hcArgRS mRNA in eukaryotic cells.     

Molecular Modeling and Site-Directed Mutagenesis Reveal Essential Residues for Catalysis in a Prokaryote-Type Aspartate Aminotransferase

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the “open-to-closed” transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.     

Uptake and processing of DNA by Acinetobacter calcoaceticus – a review

In natural transformation, DNA in the form of macromolecular fragments can be translocated across the cell envelope of prokaryotic microorganisms. During the past two decades, several, largely mutually contradictory, hypotheses have been forwarded to explain the molecular mechanism and bioenergetics of this translocation process. Other biomacromolecules are translocated across the bacterial cell envelope as well, such as polysaccharides and proteins, the latter for instance in the process of the assembly of type-IV pili. This brings up the question whether or not common components are involved.Here, we review analyses of DNA translocation in Acinetobacter calcoaceticus, a Gram-negative eubacterium that is able to migrate through twitching motility, and also shows a high frequency of natural transformation. DNA uptake in this organism is an energy-dependent process. Upon entry into the cells, the DNA fragments are integrated into the resident chromosome when a sufficiently large region of mutual homology is available (200 to 400 bp). However, this process is rather inefficient, and on the average 500 bp of each incoming fragment is degraded through exonuclease activity. Upon covalent attachment of a bulky protein molecule to the transforming DNA, the DNA-translocation machinery becomes blocked in further translocation activity.Since A. calcoaceticus is not well suited for transposon mutagenesis, a random mutagenesis procedure has been developed, based on the ligation of an antibiotic-resistance marker to random fragments of chromosomal DNA. This method was used to generate several mutants impaired in the natural transformation process. Three of these have been characterized in detail. No components, common to the translocation of macromolecules through the cell envelope of Acinetobacter, have been detected in this screen.     

Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA

Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in the phylogenetic tree, which is most closely related to that of RNA interference-mediating eukaryotic Argonautes. Here we describe a functional and mechanistic characterization of PfAgo. Like the bacterial counterparts, archaeal PfAgo contributes to host defense by interfering with the uptake of plasmid DNA. PfAgo utilizes small 5′-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target. Thus, with respect to function and specificity, the archaeal PfAgo resembles bacterial Argonautes much more than eukaryotic Argonautes. These findings demonstrate that the role of Argonautes is conserved through the bacterial and archaeal domains of life and suggests that eukaryotic Argonautes are derived from DNA-guided DNA-interfering host defense systems.