Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants. A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and f lagellar assembly apparatus in animal pathogenic bacteria. Here we report that Pseudomonas syringae pv. tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes. Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively. Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells. Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus. Finally, we show that a nonpolar hrpA mutant of P. syringae pv. tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants.Major groups of Gram-negative plant pathogenic bacteria belonging to genera Erwinia, Pseudomonas, Ralstonia, and Xanthomonas contain hypersensitive reaction and pathogenicity (hrp) genes. These genes control the ability of these bacteria to initiate interactions with plants, including elicitation of the hypersensitive reaction (HR), characterized by rapid localized death of plant cells at the pathogen infection site in resistant plants and causation of disease in susceptible plants (1, 2).hrp genes of Pseudomonas syringae are expressed in planta as a result of a regulatory cascade involving the gene products of hrpS and hrpR, positive transcriptional regulators, and of hrpL, an alternative sigma factor (3, 4). HrpL recognizes a consensus sequence motif (''harp box'') that has been identified in the upstream regions of many hrp and avr genes (4). The expression of hrp genes of many P. syringae pathovars can also be induced in vitro when bacteria are grown in defined minimal medium with low pH and containing certain sugars or sugar alcohols as carbon sources (5-7).The 25-kb hrp͞hrmA gene cluster of Pseudomonas syringae pv. syringae strain 61 is sufficient to enable nonpathogenic strains of Pseudomonas fluorescens and Escherichia coli to elicit the HR in nonhost plants (8). Sixteen of the 25 genes in this completely sequenced hrp͞hrmA gene cluster are either predicted or shown to be required for secretion of harpin Pss , a proteinaceous elicitor of the HR encoded by hrpZ (9, 10). Nine of these hrp genes, recently renamed hrc genes (11), are broadly conserved among P. syringae pathovars, Erwi...

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 x 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.

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.

Metagenomic studies have increased the amount of information on the nucleotide sequence space in our environment. It has also increased our awareness of the viral abundance and diversity not recognized before (16,24,26). Along with this new information, we have learned to acknowledge the significance of viruses in the evolution and behavior of other organisms (55

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 ␤-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.archaeal virus ͉ electron cryomicroscopy ͉ infection ͉ symmetry mismatch ͉ capsid protein evolution B ecause of the high mutation rates in viruses, amino acid sequence-analysis methods generally only reveal the relationships between closely related viruses. Instead, viral evolutionary links can be studied by using conserved structural information; for example, a tentative viral lineage has been constructed by analysis of the structures of the major capsid proteins (MCPs) of adenoviruses (infect mammals and birds), Paramecium bursaria Chlorella virus type 1 (infects unicellular algae) (1), and bacteriophages PRD1 (infects Gram-negative bacteria) and Bam35 (infects Gram-positive bacteria). The MCPs of all these viruses are trimers of double ␤-barrels in which the axis of the barrel is oriented normal to the capsid shell (2-4). Could this tentative viral lineage be expanded further? The pool of structural information on viruses that infect eukaryotic and bacterial hosts is relatively large and constantly growing, but our knowledge about viruses that infect Archaea is still very limited. The only three-dimensional structure available at 27-Å resolution is that of the Sulfolobus turreted icosahedral virus (STIV) (5), which infects Sulfolobus solfataricus in the archaeal kingdom Crenarchaeota. As the name suggests, the STIV capsid is icosahedral and has elaborate turret-like structures at the fivefold vertices (5). Its glycosylated MCP (6) also has a double ␤-barrel fold (7), suggesting a common ancestry with the PRD1-adenovirus lineage.SH1 is a virus that infects the halophilic organism Haloarcula hispanica in the archaeal kingdom of the Euryarchaeota (8). Its linear dsDNA genome of 30,898 bp has 56 identified ORFs (9). It has been shown to have several lipid moieties and 11 structural protein species. The proteins have been classified into capsid proteins and lipid core proteins (found with the membrane vesicle enclosing the nucleic acid) by biochemical analysis of virion dissociation products (10). The most abundant proteins are the capsid-associated proteins VP3, VP4, and VP7 and the lipid core protein VP12. The viral membrane is composed of neutral lipids and three major archaeal phosp...

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Archaeal viruses have been the subject of recent interest due to the diversity discovered in their virion architectures. Recently, a new group of haloarchaeal pleomorphic viruses has been discovered. It is distinctive in terms of the virion morphology and different genome types (ssDNA/dsDNA) harboured by rather closely related representatives. To date there are seven isolated viruses belonging to this group. Most of these share a cluster of five conserved genes, two of which encode major structural proteins. Putative proviruses and proviral remnants containing homologues of the conserved gene cluster were also identified suggesting a long-standing relationship of these viruses with their hosts. Comparative genomic analysis revealed three different ways of the genome organization, which possibly reflect different replication strategies employed by these viruses. The dsDNA genomes of two of these viruses were shown to contain single-strand interruptions. Further studies on one of the genomes suggested that the interruptions are located along the genome in a sequence-specific manner and exhibit polarity in distribution.

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 singlestranded 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.