Autoselection of cytoplasmic yeast virus like elements encoding toxin/antitoxin systems involves a nuclear barrier for immunity gene expression.

Cytoplasmic virus like elements (VLEs) from Kluyveromyces lactis (Kl), Pichia acaciae (Pa) and Debaryomyces robertsiae (Dr) are extremely A/T-rich (>75%) and encode toxic anticodon nucleases (ACNases) along with specific immunity proteins. Here we show that nuclear, not cytoplasmic expression of either immunity gene (PaORF4, KlORF3 or DrORF5) results in transcript fragmentation and is insufficient to establish immunity to the cognate ACNase. Since rapid amplification of 3' ends (RACE) as well as linker ligation of immunity transcripts expressed in the nucleus revealed polyadenylation to occur along with fragmentation, ORF-internal poly(A) site cleavage due to the high A/T content is likely to prevent functional expression of the immunity genes. Consistently, lowering the A/T content of PaORF4 to 55% and KlORF3 to 46% by gene synthesis entirely prevented transcript cleavage and permitted functional nuclear expression leading to full immunity against the respective ACNase toxin. Consistent with a specific adaptation of the immunity proteins to the cognate ACNases, cross-immunity to non-cognate ACNases is neither conferred by PaOrf4 nor KlOrf3. Thus, the high A/T content of cytoplasmic VLEs minimizes the potential of functional nuclear recruitment of VLE encoded genes, in particular those involved in autoselection of the VLEs via a toxin/antitoxin principle.

DNA damage induced by the anticodon nuclease from a Pichia acaciae killer strain is linked to ribonucleotide reductase depletion.

Virus like element (VLE) encoded killer toxins of Pichia acaciae and Kluyveromyces lactis kill target cells through anticodon nuclease (ACNase) activity directed against tRNA(Gln) and tRNA(Glu) respectively. Not only does tRNA cleavage disable translation, it also affects DNA integrity as well. Consistent with DNA damage, which is involved in toxicity, target cells' mutation frequencies are elevated upon ACNase exposure, suggesting a link between translational integrity and genome surveillance. Here, we analysed whether ACNase action impedes the periodically and highly expressed S-phase specific ribonucleotide reductase (RNR) and proved that RNR expression is severely affected by PaT. Because RNR catalyses the rate-limiting step in dNTP synthesis, mutants affected in dNTP synthesis were scrutinized with respect to ACNase action. Mutations elevating cellular dNTPs antagonized the action of both the above ACNases, whereas mutations lowering dNTPs aggravated toxicity. Consistently, prevention of tRNA cleavage in elp3 or trm9 mutants, which both affect the wobble uridine modification of the target tRNA, suppressed the toxin hypersensitivity of a dNTP synthesis mutant. Moreover, dNTP synthesis defects exacerbated the PaT ACNase sensitivity of cells defective in homologous recombination, proving that dNTP depletion is responsible for subsequent DNA damage.

The restriction modification system of Bacillus licheniformis MS1 and generation of a readily transformable deletion mutant.

Restriction modification systems (R-M systems), consisting of a restriction endonuclease and a cognate methyltransferase, constitute an effective means of a cell to protect itself from foreign DNA. Identification, characterization, and deletion of the restriction modification system BliMSI, a putative isoschizomer of ClaI from Caryophanon latum, were performed in the wild isolate Bacillus licheniformis MS1. BliMSI was produced as recombinant protein in Escherichia coli, purified, and in vitro analysis demonstrated identical restriction endonuclease activity as for ClaI. A recombinant E. coli strain, expressing the heterologous bliMSIM gene, was constructed and used as the host for in vivo methylation of plasmids prior to their introduction into B. licheniformis to improve transformation efficiencies. The establishment of suicide plasmids in the latter was rendered possible. The subsequent deletion of the restriction endonuclease encoding gene, bliMSIR, caused doubled transformation efficiencies in the respective mutant B. licheniformis MS2 (∆bliMSIR). Along with above in vivo methylation, the establishment of further gene deletions (∆upp, ∆yqfD) was performed. The constructed triple mutant (∆bliMSIR, ∆upp, ∆yqfD) enables rapid genome manipulation, a requirement for genetic engineering of industrially important strains.

rRNA fragmentation induced by a yeast killer toxin.

Virus like dsDNA elements (VLE) in yeast were previously shown to encode the killer toxins PaT and zymocin, which target distinct tRNA species via specific anticodon nuclease (ACNase) activities. Here, we characterize a third member of the VLE-encoded toxins, PiT from Pichia inositovora, and identify PiOrf4 as the cytotoxic subunit by conditional expression in Saccharomyces cerevisiae. In contrast to the tRNA targeting toxins, however, neither a change of the wobble uridine modification status by introduction of elp3 or trm9 mutations nor tRNA overexpression rescued from PiOrf4 toxicity. Consistent with a distinct RNA target, expression of PiOrf4 causes specific fragmentation of the 25S and 18S rRNA. A stable cleavage product comprising the first ∼ 130 nucleotides of the 18S rRNA was purified and characterized by linker ligation and subsequent reverse transcription; 3'-termini were mapped to nucleotide 131 and 132 of the 18S rRNA sequence, a region showing some similarity to the anticodon loop of tRNA(Glu)(UUC), the zymocin target. PiOrf4 residues Glu9 and His214, corresponding to catalytic sites Glu9 and His209 in the ACNase subunit of zymocin are essential for in vivo toxicity and rRNA fragmentation, raising the possibility of functionally conserved RNase modules in both proteins.

What renders Bacilli genetically competent? A gaze beyond the model organism.

Natural genetic competence enables bacteria to take in and establish exogenously supplied DNA and thus constitutes a valuable tool for strain improvement. Extensively studied in the Gram-positive model organism Bacillus subtilis genetic competence has indeed proven successful for genetic manipulation aiming at enhancement of handling, yield, and biosafety. The majority of Bacilli, particularly those relevant for industrial application, do not or only poorly develop genetic competence, although rather homologous DNA-uptake machineries are routinely encoded. Establishing the competent state solely due to high cell densities (quorum sensing dependency) appears to be restricted to the model organism, in which the small signalling peptide ComS initiates the regulatory pathway that ultimately leads to the expression of all genes necessary for reaching the competent state. Agreeing with the lack of a functional ComS peptide, competence-mediated transformation of other Bacilli depends on nutrient exhaustion rather than cell density. Genetically, competent strains of the model organism B. subtilis, cultivated for a long time and selected for laboratory purposes, display probably not least to such selection a point mutation in the promoter of a regulatory gene that favors competence development whereas the wild-type progenitor only poorly displays genetic competence. Consistent with competence being a matter of deregulation, all strains of Bacillus licheniformis displaying efficient DNA uptake were found to carry mutations in regulator genes, which are responsible for their genetic competence. Thus, strain-specific genetic equipment and regulation as well as the proven role of domestication for the well-established laboratory strains ought to be considered when attempting to broaden the applicability of competence as a genetic tool for strains other than the model organism.

The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

Unravelling the genetic basis for competence development of auxotrophic Bacillus licheniformis 9945A strains.

Bacterial natural genetic competence - well studied in Bacillus subtilis - enables cells to take up and integrate extracellularly supplied DNA into their own genome. However, little is known about competence development and its regulation in other members of the genus, although DNA uptake machineries are routinely encoded. Auxotrophic Bacillus licheniformis 9945A derivatives, obtained from repeated rounds of random mutagenesis, were long known to develop natural competence. Inspection of the colony morphology and extracellular enzyme secretion of two of these derivatives, M28 and M18, suggested that regulator genes are collaterally hit. M28 emerged as a 14 bp deletion mutant concomitantly displaying a shift in the reading frame of degS that encodes the sensor histidine kinase, which is part of the molecular switch that directs cells to genetic competence, the synthesis of extracellular enzymes or biofilm formation, while for M18, sequencing of the suspected gene revealed a 375 bp deletion in abrB, encoding the major transition state regulator. With respect to colony morphology, enzyme secretion and competence development, both of the mutations, when newly generated on the wild-type B. licheniformis 9945A genetic background, resulted in phenotypes resembling M28 and M18, respectively. All of the known naturally competent B. licheniformis representatives, hitherto thoroughly investigated in this regard, carry mutations in regulator genes, and hence genetic competence observed in domesticated strains supposedly results from deregulation.

Immunity factors for two related tRNAGln targeting killer toxins distinguish cognate and non-cognate toxic subunits.

The cytoplasmic virus-like element pWR1A from Debaryomyces robertsiae encodes a toxin (DrT) with similarities to the Pichia acaciae killer toxin PaT, which acts by importing a toxin subunit (PaOrf2) with tRNA anticodon nuclease activity into target cells. As for PaT, loss of the tRNA methyltransferase Trm9 or overexpression of tRNA(Gln) increases DrT resistance and the amount of tRNA(Gln) is reduced upon toxin exposure or upon induced intracellular expression of the toxic DrT subunit gene DrORF3, indicating DrT and PaT to share the same in vivo target. Consistent with a specific tRNase activity of DrOrf3, the protein cleaves tRNA(Gln) but not tRNA(Glu) in vitro. Heterologous cytoplasmic expression identified DrOrf5 as the DrT specific immunity factor; it confers resistance to exogenous DrT as well as to intracellular expression of DrOrf3 and prevents tRNA depletion by the latter. The PaT immunity factor PaOrf4, a homologue of DrOrf5 disables intracellular action of both toxins. However, the DrT protection level mediated by PaOrf4 is reduced compared to DrOrf5, implying a recognition mechanism for the cognate toxic subunit, leading to incomplete toxicity suppression of similar, but non-cognate toxic subunits.

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair.

PaOrf2 and γ-toxin subunits of Pichia acaciae toxin (PaT) and Kluyveromyces lactis zymocin are tRNA anticodon nucleases. These secreted ribotoxins are assimilated by Saccharomyces cerevisiae, wherein they arrest growth by depleting specific tRNAs. Toxicity can be recapitulated by induced intracellular expression of PaOrf2 or γ-toxin in S. cerevisiae. Mutational analysis of γ-toxin has identified amino acids required for ribotoxicity in vivo and RNA transesterification in vitro. Here, we report that PaOrf2 residues Glu9 and His287 (putative counterparts of γ-toxin Glu9 and His209) are essential for toxicity. Our results suggest a similar basis for RNA transesterification by PaOrf2 and γ-toxin, despite their dissimilar primary structures and distinctive tRNA target specificities. PaOrf2 makes two sequential incisions in tRNA, the first of which occurs 3' from the mcm(5)s(2)U wobble nucleoside and depends on mcm(5). A second incision two nucleotides upstream results in the net excision of a di-nucleotide. Expression of phage and plant tRNA repair systems can relieve PaOrf2 toxicity when tRNA cleavage is restricted to the secondary site in elp3 cells that lack the mcm(5) wobble U modification. Whereas the endogenous yeast tRNA ligase Trl1 can heal tRNA halves produced by PaOrf2 cleavage in elp3 cells, its RNA sealing activity is inadequate to complete the repair. Compatible sealing activity can be provided in trans by plant tRNA ligase. The damage-rescuing ability of tRNA repair systems is lost when PaOrf2 can break tRNA at both sites. These results highlight the logic of a two-incision mechanism of tRNA anticodon damage that evades productive repair by tRNA ligases.

Site-directed mutagenesis of the heterotrimeric killer toxin zymocin identifies residues required for early steps in toxin action.

Zymocin is a Kluyveromyces lactis protein toxin composed of αßγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αß-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activity in vitro that might be important during γ import. Saccharomyces cerevisiae strains carrying k1-derived hybrid elements deficient in either αß (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of ß (Cys250). Since ßγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of ß C250 and γ C231 in zymocin assembly. To test the capability of αß to carry alternative cargos, the heterologous ACNase from Pichia acaciae (P. acaciae Orf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αß's capability to deliver other cargo proteins into target cells.

yneA mRNA instability is involved in temporary inhibition of cell division during the SOS response of Bacillus megaterium.

The SOS response, a mechanism enabling bacteria to cope with DNA damage, is strictly regulated by the two major players, RecA and LexA (Bacillus homologue DinR). Genetic stress provokes formation of ssDNA-RecA nucleoprotein filaments, the coprotease activity of which mediates the autocatalytic cleavage of the transcriptional repressor DinR and ensures the expression of a set of din (damage-inducible) genes, which encode proteins that enhance repair capacity, accelerate mutagenesis rate and cause inhibition of cell division (ICD). In Bacillus subtilis, the transcriptional activation of the yneAB-ynzC operon is part of the SOS response, with YneA being responsible for the ICD. Pointing to its cellular function in Bacillus megaterium, overexpression of homologous YneA led to filamentous growth, while ICD was temporary during the SOS response. Genetic knockouts of the individual open reading frames of the yneAB-ynzC operon increased the mutagenic sensitivity, proving - for the first time in a Bacillus species - that each of the three genes is in fact instrumental in coping with genetic stress. Northern- and quantitative real-time PCR analyses revealed - in contrast to other din genes (exemplified for dinR, uvrBA) - transient mRNA-presence of the yneAB-ynzC operon irrespective of persisting SOS-inducing conditions. Promoter test assays and Northern analyses suggest that the decline of the ICD is at least partly due to yneAB-ynzC mRNA instability.

Exoglucanase-encoding genes from three Wickerhamomyces anomalus killer strains isolated from olive brine.

Wickerhamomyces anomalus killer strains are important for fighting pathogenic yeasts and for controlling harmful yeasts and bacteria in the food industry. Targeted disruption of key genes in ß-glucan synthesis of a sensitive Saccharomyces cerevisiae strain conferred resistance to the toxins of W. anomalus strains BS91, BCA15 and BCU24 isolated from olive brine. Competitive inhibition of the killing activities by laminarin and pustulan refer to ß-1,3- and ß-1,6-glucans as the main primary toxin targets. The extracellular exoglucanase-encoding genes WaEXG1 and WaEXG2 from the three strains were sequenced and were found to display noticeable similarities to those from known potent W. anomalus killer strains.

Generation of biologically contained, readily transformable, and genetically manageable mutants of the biotechnologically important Bacillus pumilus.

Bacillus pumilus mutants were generated by targeted deletion of a set of genes eventually facilitating genetic handling and assuring biological containment. The well-defined and stable mutants do not form functional endospores due to the deletion of yqfD, an essential sporulation gene; they are affected in DNA repair, as ΔuvrBA rendered them UV hypersensitive and, thus, biologically contained; they are deficient for the uracil phosphoribosyl-transferase (Δupp), allowing for 5-fluorouracil-based counterselection facilitating rapid allelic exchanges; and they are readily transformable due to the deletion of the restrictase encoding locus (ΔhsdR) of a type I restriction modification system. Vegetative growth as well as extracellular enzyme production and secretion are in no case affected. The combination of such gene deletions allows for development of B. pumilus strains suited for industrial use and further improvements.

Extrachromosomal genetic elements in Micrococcus.

Micrococci are Gram-positive G + C-rich, nonmotile, nonspore-forming actinomycetous bacteria. Micrococcus comprises ten members, with Micrococcus luteus being the type species. Representatives of the genus play important roles in the biodegradation of xenobiotics, bioremediation processes, production of biotechnologically important enzymes or bioactive compounds, as test strains in biological assays for lysozyme and antibiotics, and as infective agents in immunocompromised humans. The first description of plasmids dates back approximately 28 years, when several extrachromosomal elements ranging in size from 1.5 to 30.2 kb were found in Micrococcus luteus. Up to the present, a number of circular plasmids conferring antibiotic resistance, the ability to degrade aromatic compounds, and osmotolerance are known, as well as cryptic elements with unidentified functions. Here, we review the Micrococcus extrachromosomal traits reported thus far including phages and the only quite recently described large linear extrachromosomal genetic elements, termed linear plasmids, which range in size from 75 kb (pJD12) to 110 kb (pLMA1) and which confer putative advantageous capabilities, such as antibiotic or heavy metal resistances (inferred from sequence analyses and curing experiments). The role of the extrachromosomal elements for the frequently proven ecological and biotechnological versatility of the genus will be addressed as well as their potential for the development and use as genetic tools.

Repeated capture of a cytoplasmic linear plasmid by the host nucleus in Debaryomyces hansenii.

Debaryomyces hansenii is a halotolerant yeast species that has been shown to carry various nuclear genes of plasmid or viral origin (NUPAVs). However, a recent ancestor of such NUPAVs has not been identified. Here we determined for the first time the molecular structure of an entire cytoplasmic linear plasmid, pDH1A, indigenous to this species. The element is related to non-autonomous killer plasmids from Kluyveromyces lactis and Pichia acaciae and carries a B-type DNA polymerase as well as remnants of a killer toxin system, a secreted chitin-binding protein. Other essential toxin subunits or an immunity function, however, appear to be lost, while two additional small open reading frames are present. Transcripts for all four genes located on pDH1A could be verified by RT-PCR. Interestingly, all genes from pDH1A could be identified as ancestors of NUPAVs located at different chromosomes within the nucleus of D. hansenii, suggesting repeated nuclear capture of fragments originating from pDH1A.

Anticodon nuclease encoding virus-like elements in yeast.

A variety of yeast species are known to host systems of cytoplasmic linear dsDNA molecules that establish replication and transcription independent of the nucleus via self-encoded enzymes that are phylogenetically related to those encoded by true infective viruses. Such yeast virus-like elements (VLE) fall into two categories: autonomous VLEs encode all the essential functions for their inheritance, and additional, dependent VLEs, which may encode a toxin-antitoxin system, generally referred to as killer toxin and immunity. In the two cases studied in depth, killer toxin action relies on chitin binding and hydrophobic domains, together allowing a separate toxic subunit to sneak into the target cell. Mechanistically, the latter sabotages codon-anticodon interaction by endonucleolytic cleavage of specific tRNAs 3' of the wobble nucleotide. This primary action provokes a number of downstream effects, including DNA damage accumulation, which contribute to the cell-killing efficiency and highlight the importance of proper transcript decoding capacity for other cellular processes than translation itself. Since wobble uridine modifications are crucial for efficient anticodon nuclease (ACNase) action of yeast killer toxins, the latter are valuable tools for the characterization of a surprisingly complex network regulating the addition of wobble base modifications in tRNA.

Native Cultivable Bacteria from the Blueberry Microbiome as Novel Potential Biocontrol Agents.

Blueberry production is affected by fungal postharvest pathogens, including Botrytis cinerea and Alternaria alternata, the causative agents of gray mold disease and Alternaria rot, respectively. Biocontrol agents adapted to blueberries and local environments are not known to date. Here, we report on the search for and the identification of cultivable blueberry epiphytic bacteria with the potential to combat the aforementioned fungi. Native, blueberry-borne bacterial strains were isolated from a plantation in Tucumán, Argentina and classified based on 16S rRNA gene sequences. Antagonistic activities directed at B. cinerea and A. alternata were studied in vitro and in vivo. The 22 bacterial strains obtained could be attributed to eleven different genera: Rosenbergiella, Fictibacillus, Bacillus, Pseudomonas, Microbacterium, Asaia, Acinetobacter, Curtobacterium, Serratia, Sphingomonas and Xylophilus. Three strains displaying antagonistic impacts on the fungal pathogens were identified as Bacillus velezensis (BA3 and BA4) and Asaia spathodeae (BMEF1). These strains are candidates for biological control agents of local blueberry production and might provide a basis for the development of eco-friendly, sustainable alternatives to synthetic pesticides.

DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins.

Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT) were found to disable translation in target cells by virtue of anticodon nuclease (ACNase) activities on tRNA(Glu) and tRNA(Gln), respectively. Surprisingly, however, ACNase exposure does not only impair translation, but also affects genome integrity and concomitantly DNA damage occurs. Previously, it was shown that homologous recombination protects cells from ACNase toxicity. Here, we have analyzed whether other DNA repair pathways are functional in conferring ACNase resistance as well. In addition to HR, base excision repair (BER) and postreplication repair (PRR) promote clear resistance to either, PaT and zymocin. Comparative toxin sensitivity analysis of BER mutants revealed that its ACNase protective function is due to the endonucleases acting on apurinic (AP) sites, whereas none of the known DNA glycosylases is involved. Because PaT and zymocin require the presence of the ELP3/TRM9-dependent wobble uridine modification 5-methoxy-carbonyl-methyl (mcm(5)) for tRNA cleavage, we analyzed toxin response in DNA repair mutants additionally lacking such tRNA modifications. ACNase resistance caused by elp3 or trm9 mutations was found to rescue hypersensitivity of DNA repair defects, consistent with DNA damage to occur as a consequence of tRNA cleavage. The obtained genetic evidence promises to reveal new aspects into the mechanism linking translational fidelity and genome surveillance.

Genetic control of amadori product degradation in Bacillus subtilis via regulation of frlBONMD expression by FrlR.

Bacillus subtilis is capable of degrading fructosamines. The phosphorylation and the cleavage of the resulting fructosamine 6-phosphates is catalyzed by the frlD and frlB gene products, respectively. This study addresses the physiological importance of the frlBONMD genes (formerly yurPONML), revealing the necessity of their expression for growth on fructosamines and focusing on the complex regulation of the corresponding transcription unit. In addition to the known regulation by the global transcriptional regulator CodY, the frl genes are repressed by the convergently transcribed FrlR (formerly YurK). The latter causes repression during growth on substrates other than fructosamines. Additionally, we identified in the first intergenic region of the operon an FrlR binding site which is centrally located within a 38-bp perfect palindromic sequence. There is genetic evidence that this sequence, in combination with FrlR, contributes to the remarkable decrease in the transcription downstream of the first gene of the frl operon.

Replication-involved genes of pAL1, the linear plasmid of Arthrobacter nitroguajacolicus Rü61a--phylogenetic and transcriptional analysis.

The 113-kb pAL1 is the only Arthrobacter linear plasmid known; it has terminal inverted repeats and 5' covalently attached terminal proteins (TPs). The latter and a telomere-associated protein (Tap) are encoded by plasmid ORFs 102 and 101, respectively. As for Streptomyces linear replicons, in which both above proteins are instrumental in telomere patching, they are involved in pAL1 replication as well. However, the alignment of actinobacterial Taps and TPs revealed that pAL1 and the linear elements from Rhodococci comprise a discrete phylogenetic group, clearly delineated from the streptomycetes linear plasmids. In line with such findings is the same genetic arrangement of ORF 101 and 102 counterparts in the rhodococcal elements. Furthermore, the adjacent gene (ORF100) has matches in the rhodococcal plasmids as well. In linear elements of Streptomyces there is no ORF100 homolog. Two alternative annotations are possible for ORF100 gene products. As RT-PCR revealed cotranscription of ORFs 100-102, the ORF100 gene product is presumably involved in replicative processes. Taken also into consideration the likely absence of an internal replication origin (other than in Streptomyces linear elements), we assume a distinct replication/telomere patching mechanism for pAL1 type replicons.