Tamara Basta

Detection and Characterization of Conjugative Degradative Plasmids in Xenobiotic-Degrading Sphingomonas Strains

A systematic survey for the presence of plasmids in 17 different xenobiotic-degrading Sphingomonas strains was performed. In almost all analyzed strains, two to five plasmids with sizes of about 50 to 500 kb were detected by using pulsed-field gel electrophoresis. A comparison of plasmid preparations untreated or treated with S1 nuclease suggested that, in general, Sphingomonas plasmids are circular. Hybridization experiments with labeled gene probes suggested that large plasmids are involved in the degradation of dibenzo-p-dioxin, dibenzofuran, and naphthalenesulfonates in S. wittichii RW1, Sphingomonas sp. HH69, and S. xenophaga BN6, respectively. The plasmids which are responsible for the degradation of naphthalene, biphenyl, and toluene by S. aromaticivorans F199 (pNL1) and of naphthalenesulfonates by S. xenophaga BN6 (pBN6) were site-specifically labeled with a kanamycin resistance cassette. The conjugative transfer of these labeled plasmids was attempted with various bacterial strains as putative recipient strains. Thus, a conjugative transfer of plasmid pBN6 from S. xenophaga BN6 to a cured mutant of strain BN6 and to Sphingomonas sp. SS3 was observed. The conjugation experiments with plasmid pNL1 suggested a broader host range of this plasmid, because it was transferred without any obvious structural changes to S. yanoikuyae B1, Sphingomonas sp. SS3, and S. herbicidovorans. In contrast, major plasmid rearrangements were observed in the transconjugants after the transfer of plasmid pNL1 to Sphingomonas sp. HH69 and of pBN6 to Sphingomonas sp. SS3. No indications for the transfer of a Sphingomonas plasmid to bacteria outside of the Sphingomonadaceae were obtained.

Structure of the Acidianus Filamentous Virus 3 and Comparative Genomics of Related Archaeal Lipothrixviruses

ABSTRACT Four novel filamentous viruses with double-stranded DNA genomes, namely, Acidianus filamentous virus 3 (AFV3), AFV6, AFV7, and AFV8, have been characterized from the hyperthermophilic archaeal genus Acidianus, and they are assigned to the Betalipothrixvirus genus of the family Lipothrixviridae. The structures of the approximately 2-μm-long virions are similar, and one of them, AFV3, was studied in detail. It consists of a cylindrical envelope containing globular subunits arranged in a helical formation that is unique for any known double-stranded DNA virus. The envelope is 3.1 nm thick and encases an inner core with two parallel rows of protein subunits arranged like a zipper. Each end of the virion is tapered and carries three short filaments. Two major structural proteins were identified as being common to all betalipothrixviruses. The viral genomes were sequenced and analyzed, and they reveal a high level of conservation in both gene content and gene order over large regions, with this similarity extending partly to the earlier described betalipothrixvirus Sulfolobus islandicus filamentous virus. A few predicted gene products of each virus, in addition to the structural proteins, could be assigned specific functions, including a putative helicase involved in Holliday junction branch migration, a nuclease, a protein phosphatase, transcriptional regulators, and glycosyltransferases. The AFV7 genome appears to have undergone intergenomic recombination with a large section of an AFV2-like viral genome, apparently resulting in phenotypic changes, as revealed by the presence of AFV2-like termini in the AFV7 virions. Shared features of the genomes include (i) large inverted terminal repeats exhibiting conserved, regularly spaced direct repeats; (ii) a highly conserved operon encoding the two major structural proteins; (iii) multiple overlapping open reading frames, which may be indicative of gene recoding; (iv) putative 12-bp genetic elements; and (v) partial gene sequences corresponding closely to spacer sequences of chromosomal repeat clusters.

Novel archaeal plasmid pAH1 and its interactions with the lipothrixvirus AFV1

At present very little is known about interactions between extrachromosomal genetic elements in Archaea. Here we describe an Acidianus strain which carries naturally a novel 28 kb conjugative plasmid‐like element, pAH1, and also serves as a laboratory host for lipothrixvirus AFV1. In an attempt to establish a system for studying plasmid–virus interactions we characterized the genome of pAH1 which closely resembles those of the Sulfolobus conjugative plasmids pARN3 and pARN4. pAH1 integrates site specifically into, and excises from, the host chromosome indicating a dynamic interaction with the latter. Although nucleotide sequence comparisons revealed extensive intergenomic exchange during the evolution of archaeal conjugative plasmids, pAH1 was shown to be stably maintained suggesting that the host system is suitable for studying plasmid–virus interactions. AFV1 infection and propagation leads to a loss of the circular form of pAH1 and this effect correlates positively with the increase in the intracellular quantity of AFV1 DNA. We infer that the virus inhibits plasmid replication since no pAH1 degradation was observed. This mechanism of archaeal viral inhibition of plasmid propagation is not observed in bacteria where relevant bacteriophages either are dependent on a conjugative plasmid for successful infection or are excluded by a resident plasmid.

The thermo- and acido-stable ORF-99 from the archaeal virus AFV1.

Acidianus Filamentous Virus 1 (AFV1), isolated from acidic hot springs, is an enveloped lipid‐containing archaeal filamentous virus with a linear double‐stranded DNA genome. It infects Acidianus, which is a hyperthermostable archaea growing at 85°C and acidic pHs, below pH 3. AFV1‐99, a protein of 99 amino acids of unknown function, has homologues in the archaeal virus families Lipothrixviridae and Rudiviridae. We determined the crystal structure of AFV1‐99 at 2.05 Å resolution. AFV1‐99 has a new fold, is hyperthermostable (up to 95°C) and resists to extreme pH (between pH 0 and 11) and to the combination of high temperature (95°C) and low pH (pH 0). It possesses characteristics of hyperthermostable proteins, such as a high content of charged residues.

Unique genome replication mechanism of the archaeal virus AFV1.

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two‐dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D‐loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark‐field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.

The crystal structure of ORF14 from Sulfolobus islandicus filamentous virus.

Studies on viruses in hot aquatic ecosystems, at temperatures above 808C, revealed unusual morphotypes and quasi-orphan genome sequences which made it possible to establish several new families of double-stranded (ds) DNA viruses. Among them, the Lipothrixviridae are enveloped lipid-containing filamentous viruses with linear double-stranded DNA genomes (AFV1-8, TTV1-3, and Sulfolobus islandicus filamentous virus [SIFV]).1–4 The hosts of these viruses are hyperthermophilic and hyperacidophilic archaea of the genera Acidianus or Sulfolobus. The 35 genomes of archaeal viruses that are presently sequenced have in common that more than 80% of their ORFs do not share any sequence homology with ORFs of other viruses or organisms apart from other archaeal viruses.4 Putative functions based on weak sequence identities have been proposed for a few ORFs, including structural proteins, glycosyl transerases, helicases, and integrases. Apart from structural proteins, it is expected that other ORFs should code for enzymes or proteins involved in viral infection and replication, and be functional homologues of bacterial, archaeal, or eukaryotic proteins. Moreover, they may reveal novel features which can be exploited for biotechnological applications. To get insight into the function of the Lipothrixviridae proteome, we have initiated a program to determine the structure of conserved ORFs. We present here the crystal structure of ORF14 of SIFV (Lipothrixviridae), a protein of 112 residues which has homologues in other archaeal viruses, Sulfolobus islandicus rod-shaped virus 1 (SIRV1, Rudiviridae; ORF99, ORF100, ORF95, and ORF96) and Acidianus filamentous virus 1 (AFV1, Lipothrixviridae; ORF99) [Fig. 1(A)]. SIFV-14 has a domain swapped structure and forms fiber structures in the crystal.

Structure and function of the 3-carboxy-cis,cis-muconate lactonizing enzyme from the protocatechuate degradative pathway of Agrobacterium radiobacter S2.

3‐carboxy‐cis,cis‐muconate lactonizing enzymes participate in the protocatechuate branch of the 3‐oxoadipate pathway of various aerobic bacteria. The gene encoding a 3‐carboxy‐cis,cis‐muconate lactonizing enzyme (pcaB1S2) was cloned from a gene cluster involved in protocatechuate degradation by Agrobacterium radiobacter strain S2. This gene encoded for a 3‐carboxy‐cis,cis‐muconate lactonizing enzyme of 353 amino acids − significantly smaller than all previously studied 3‐carboxy‐cis,cis‐muconate lactonizing enzymes. This enzyme, ArCMLE1, was produced in Escherichia coli and shown to convert not only 3‐carboxy‐cis,cis‐muconate but also 3‐sulfomuconate. ArCMLE1 was purified as a His‐tagged enzyme variant, and the basic catalytic constants for the conversion of 3‐carboxy‐cis,cis‐muconate and 3‐sulfomuconate were determined. In contrast, Agrobacterium tumefaciens 3‐carboxy‐cis,cis‐muconate lactonizing enzyme 1 could not, despite 87% sequence identity to ArCMLE1, use 3‐sulfomuconate as substrate. The crystal structure of ArCMLE1 was determined at 2.2 Å resolution. Consistent with the sequence, it showed that the C‐terminal domain, present in all other members of the fumarase II family, is missing in ArCMLE1. Nonetheless, both the tertiary and quaternary structures, and the structure of the active site, are similar to those of Pseudomonas putida 3‐carboxy‐cis,cis‐muconate lactonizing enzyme. One principal difference is that ArCMLE1 contains an Arg, as opposed to a Trp, in the active site. This indicates that activation of the carboxylic nucleophile by a hydrophobic environment is not required for lactonization, unlike earlier proposals [Yang J, Wang Y, Woolridge EM, Arora V, Petsko GA, Kozarich JW & Ringe D (2004) Biochemistry43, 10424–10434]. We identified citrate and isocitrate as noncompetitive inhibitors of ArCMLE1, and found a potential binding pocket for them on the enzyme outside the active site.

Crystallization and preliminary X‐ray diffraction analysis of protein 14 from Sulfolobus islandicus filamentous virus (SIFV)

A large-scale programme has been embarked upon aiming towards the structural determination of conserved proteins from viruses infecting hyperthermophilic archaea. Here, the crystallization of protein 14 from the archaeal virus SIFV is reported. This protein, which contains 111 residues (MW 13 465 Da), was cloned and expressed in Escherichia coli with an N-terminal His(6) tag and purified to homogeneity. The tag was subsequently cleaved and the protein was crystallized using PEG 1000 or PEG 4000 as a precipitant. Large crystals were obtained of the native and the selenomethionine-labelled protein using sitting drops of 100-300 nl. Crystals belong to space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 68.1, c = 132.4 A. Diffraction data were collected to a maximum acceptable resolution of 2.95 and 3.20 A for the SeMet-labelled and native protein, respectively.