Elina Roine

An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope

Archaeal organisms are generally known as diverse extremophiles, but they play a crucial role also in moderate environments. So far, only about 50 archaeal viruses have been described in some detail. Despite this, unusual viral morphotypes within this group have been reported. Interestingly, all isolated archaeal viruses have a double‐stranded DNA (dsDNA) genome. To further characterize the diversity of archaeal viruses, we screened highly saline water samples for archaea and their viruses. Here, we describe a new haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV‐1) that was isolated from a solar saltern and infects an indigenous host belonging to the genus Halorubrum. Infection does not cause cell lysis, but slightly retards growth of the host and results in high replication of the virus. The sequenced genome (7048 nucleotides) of HRPV‐1 is single‐stranded DNA (ssDNA), which makes HRPV‐1 the first characterized archaeal virus that does not have a dsDNA genome. In spite of this, similarities to another archaeal virus were observed. Two major structural proteins were recognized in protein analyses, and by lipid analyses it was shown that the virion contains a membrane. Electron microscopy studies indicate that the enveloped virion is pleomorphic (approximately 44 × 55 nm). HRPV‐1 virion may represent commonly used virion architecture, and it seems that structure‐based virus lineages may be extended to non‐icosahedral viruses.

Global network of specific virus-host interactions in hypersaline environments.

Hypersaline environments are dominated by archaea and bacteria and are almost entirely devoid of eukaryotic organisms. In addition, hypersaline environments contain considerable numbers of viruses. Currently, there is only a limited amount of information about these haloviruses. The ones described in detail mostly resemble head-tail bacteriophages, whereas observations based on direct microscopy of the hypersaline environmental samples highlight the abundance of non-tailed virus-like particles. Here we studied nine spatially distant hypersaline environments for the isolation of new halophilic archaea (61 isolates), halophilic bacteria (24 isolates) and their viruses (49 isolates) using a culture-dependent approach. The obtained virus isolates approximately double the number of currently described archaeal viruses. The new isolates could be divided into three tailed and two non-tailed virus morphotypes, suggesting that both types of viruses are widely distributed and characteristic for haloarchaeal viruses. We determined the sensitivity of the hosts against all isolated viruses. It appeared that the host ranges of numerous viruses extend to hosts in distant locations, supporting the idea that there is a global exchange of microbes and their viruses. It suggests that hypersaline environments worldwide function like a single habitat.

Structure and host-cell interaction of SH1, a membrane-containing, halophilic euryarchaeal virus

The Archaea, and the viruses that infect them, are the least well understood of all of the three domains of life. They often grow in extreme conditions such as hypersaline lakes and sulfuric hot springs. Only rare glimpses have been gained into the structures of archaeal viruses. Here, we report the subnanometer resolution structure of a recently isolated, hypersalinic, membrane-containing, euryarchaeal virus, SH1, in which different viral proteins can be localized. The results indicate that SH1 has a complex capsid formed from single β-barrels, an important missing link in hypotheses on viral capsid protein evolution. Unusual, symmetry-mismatched spikes seem to play a role in host adsorption. They are connected to highly organized membrane proteins providing a platform for capsid assembly and potential machinery for host infection.

Immunocytochemical localization of HrpA and HrpZ supports a role for the Hrp pilus in the transfer of effector proteins from Pseudomonas syringae pv. tomato across the host plant cell wall

The Hrp pilus, composed of HrpA subunits, is an essential component of the type III secretion system in Pseudomonas syringae. We used electron microscopy (EM) and immunocytochemistry to examine production of the pilus in vitro from P. syringae pv. tomato strain DC3000 grown under hrp-inducing conditions on EM grids. Pili, when labeled with antibodies to HrpA, developed rapidly in a nonpolar manner shortly after the detection of the hrpA transcript and extended up to 5 microm into surrounding media. Structures at the base of the pilus were clearly differentiated from the basal bodies of flagella. The HrpZ protein, also secreted via the type III system, was found by immunogold labeling to be associated with the pilus in vitro. Accumulation and secretion of HrpA and HrpZ were also examined quantitatively after the inoculation of wild-type DC3000 and hrpA and hrpZ mutants into leaves of Arabidopsis thaliana. The functional pilus crossed the plant cell wall to generate tracks of immunogold labeling for HrpA and HrpZ. Mutants that produced HrpA but did not assemble pili were nonpathogenic, did not secrete HrpA protein, and were compromised for the accumulation of HrpZ. A model is proposed in which the rapidly elongating Hrp pilus acts as a moving conveyor, facilitating transfer of effector proteins from bacteria to the plant cytoplasm across the formidable barrier of the plant cell wall.

Characterization of Type IV Pilus Genes in Pseudomonas syringae pv. tomato DC3000

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.

New, Closely Related Haloarchaeal Viral Elements with Different Nucleic Acid Types

ABSTRACT During the search for haloarchaeal viruses, we isolated and characterized a new pleomorphic lipid-containing virus, Haloarcula hispanica pleomorphic virus 1 (HHPV-1), that infects the halophilic archaeon Haloarcula hispanica. The virus contains a circular double-stranded DNA genome of 8,082 bp in size. The organization of the genome shows remarkable synteny and amino acid sequence similarity to the genome and predicted proteins of the halovirus HRPV-1, a pleomorphic single-stranded DNA virus that infects a halophilic archaeon Halorubrum sp. Analysis of the two halovirus sequences, as well as the entire nucleotide sequence of the 10.8-kb pHK2-plasmid and a 12.6-kb chromosomal region in Haloferax volcanii, allows us to suggest a new group of closely related viruses with genomes of either single-stranded or double-stranded DNA. Currently, closely related viruses are considered to have the same genome type. Our observation clearly contradicts this categorization and indicates that we should reconsider the way we classify viruses. Our results also provide a new example of related viruses where the viral structural proteins have not diverged as much as the proteins associated with genome replication. This result further strengthens the proposal for higher-order classification to be based on virion architecture rather than on genome type or replication mechanism.

MUTATIONAL ANALYSIS OF THE PSEUDOMONAS SYRINGAE PV. TOMATO HRPA GENE ENCODING HRP PILUS SUBUNIT

Plant pathogenic Pseudomonas syringae strains harbour a type III secretion pathway suggested to be involved in the delivery of effector proteins from the bacteria into plant cells. During plant interaction, the bacteria apparently produce surface appendages, termed Hrp pili, that are indispensable for the secretion process. We have created an insertion mutation library, as well as deletion mutations to hrpA, the structural gene encoding Hrp pilin. Analysis of the mutants revealed gene regions important for hrpA expression, pilus assembly and pilus‐dependent autoagglutination of the bacteria. The majority of insertions in the amino‐terminal half of the pilin were tolerated without bacterial interaction with plants being affected, while the carboxy‐terminus appeared to be needed for pilus assembly. Insertions in the 5′ non‐translated region and the first codons within the open reading frame affected mRNA production or stability and abolished protein production.

Localization of hrpA‐induced Pseudomonas syringae pv. tomato DC3000 in infected tomato leaves

SUMMARY Pseudomonas syringae pv. tomato is the causative agent of bacterial speck of tomato. The key virulence determinant of P. syringae is the hrp gene cluster, which encodes a type III secretion system. The type III system is used by a wide variety of pathogenic bacteria for transporting virulence proteins from the bacteria directly into the eukaryotic host cell. Hrp pilus, which is composed of HrpA pilin subunits, is an indispensable component of the type III secretion system in P. syringae. Here we have determined the spatial and temporal expression pattern of hrpA of P. syringae DC3000 in intact leaves, using a HrpA-GFP protein fusion and confocal microscopy. The hrpA gene was strongly and rapidly induced inside the leaf tissues after infiltration of the bacteria. After spray-inoculation, hrpA-induced bacteria were detected endophytically 72 h post-inoculation, and 96 h after spray-inoculation, disease symptoms appeared and GFP-expressing bacteria were observed at symptom sites, both endo- and epiphytically. Live/dead staining of the bacteria showed that Pst DC3000 does not survive well on leaf surfaces. Apoplastic populations were apparently bursting on to the leaf surface through stomata. Kinetics of population sizes of wild-type DC3000 and hrpA(-) showed significant differences, initially endophytically and only later epiphytically. Our results suggest that the Hrp pilus is first induced in the apoplast and apparently functions mainly inside the leaf tissues. These results suggest that P. syringae DC3000 mainly multiplies endophytically.

The Single-Stranded DNA Genome of Novel Archaeal Virus Halorubrum Pleomorphic Virus 1 Is Enclosed in the Envelope Decorated with Glycoprotein Spikes

ABSTRACT Only a few archaeal viruses have been subjected to detailed structural analyses. Major obstacles have been the extreme conditions such as high salinity or temperature needed for the propagation of these viruses. In addition, unusual morphotypes of many archaeal viruses have made it difficult to obtain further information on virion architectures. We used controlled virion dissociation to reveal the structural organization of Halorubrum pleomorphic virus 1 (HRPV-1) infecting an extremely halophilic archaeal host. The single-stranded DNA genome is enclosed in a pleomorphic membrane vesicle without detected nucleoproteins. VP4, the larger major structural protein of HRPV-1, forms glycosylated spikes on the virion surface and VP3, the smaller major structural protein, resides on the inner surface of the membrane vesicle. Together, these proteins organize the structure of the membrane vesicle. Quantitative lipid comparison of HRPV-1 and its host Halorubrum sp. revealed that HRPV-1 acquires lipids nonselectively from the host cell membrane, which is typical of pleomorphic enveloped viruses.

Hcp2, a secreted protein of the phytopathogen Pseudomonas syringae pv. tomato DC3000, is required for fitness for competition against bacteria and yeasts.

When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.