Inhibition of adenovirus replication by CRISPR-Cas9-mediated targeting of the viral E1A gene.

DNA-targeting CRISPR-Cas systems are able to cleave dsDNA in mammalian cells. Accordingly, they have been employed to target the genomes of dsDNA viruses, mostly when present in cells in a non-replicative state with low copy numbers. However, the sheer amount of viral DNA produced within a very short time by certain lytically replicating viruses potentially brings the capacities of CRISPR-Cas systems to their limits. The accessibility of viral DNA replication sites, short time of accessibility of the DNA before encapsidation, or its complexation with shielding proteins are further potential hurdles. Adenoviruses are fast-replicating dsDNA viruses for which no approved antiviral therapy currently exists. We evaluated the potency of CRISPR-Cas9 in inhibiting the replication of human adenovirus 5 in vitro by targeting its master regulator E1A with a set of guide RNAs and observed a decrease in infectious virus particles by up to three orders of magnitude. Target DNA cleavage also negatively impacted the amount of viral DNA accumulated during the infection cycle. This outcome was mainly caused by specific deletions, inversions, and duplications occurring between target sites, which abolished most E1A functions in most cases. Additionally, we compared two strategies for multiplex gRNA expression and obtained comparable results.

Bacterial-like nitric oxide synthase in the haloalkaliphilic archaeon Natronomonas pharaonis.

Bacterial nitric oxide (NO) synthases (bNOS) play diverse and important roles in microbial physiology, stress resistance, and virulence. Although bacterial and mammalian NOS enzymes have been well-characterized, comparatively little is known about the prevalence and function of NOS enzymes in Archaea. Analysis of archaeal genomes revealed that highly conserved bNOS homologs were restricted to members of the Halobacteria. Of these, Natronomonas pharaonis NOS (npNOS) was chosen for further characterization. NO production was confirmed in heterologously expressed His-tagged npNOS by coupling nitrite production from N-hydroxy-L-arginine in an H2O2-supported reaction. Additionally, the nos gene was successfully targeted and disrupted to create a Nmn. pharaonis nos mutant by adapting an established Natrialba magadii transformation protocol. Genome re-sequencing of this mutant revealed an additional frameshift in a putative cation-acetate symporter gene, which could contribute to altered acetate metabolism in the nos mutant. Inactivation of Nmn. pharaonis nos was also associated with several phenotypes congruent with bacterial nos mutants (altered growth, increased oxygen consumption, increased pigment, increased UV susceptibility), suggesting that NOS function may be conserved between bacteria and archaea. These studies are the first to describe genetic inactivation and characterization of a Nmn. pharaonis gene and provides enhanced tools for probing its physiology.

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ.

Ice nucleation (IN) active bacteria such as Pseudomonas syringae promote the growth of ice crystals more effectively than any material known. Using the specialized ice nucleation protein (INP) InaZ, P. syringae-the well studied epiphytic plant pathogen-attacks plants by frost damage and, likewise fascinating, drives ice nucleation within clouds when airborne in the atmosphere by linkage to the Earth's water cycle. While ice nucleation proteins play a tremendous role for life on the planet, the molecular details of their activity on the bacterial membrane surface are largely unknown. Bacterial ghosts (BGs) derived from Escherichia coli can be used as simplified model systems to study the mode of action of InaZ. In this work, the authors used BGs to study the role of InaZ localization on the luminal side of the bacterial inner membrane. Naturally, P. syringae INPs are displayed on the surface of the outer membrane; so in contrast, the authors engineered an N-terminal truncated form of inaZ lacking the transport sequence for anchoring of InaZ on the outer membrane. This construct was fused to N- and C-terminal inner membrane anchors and expressed in Escherichia coli C41. The IN activity of the corresponding living recombinant E. coli catalyzing interfacial ice formation of supercooled water at high subzero temperatures was tested by a droplet-freezing assay and surface spectroscopy. The median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at -20.1 °C and with inner membrane anchored INPs at a T50 value between -7 and -9 °C, demonstrating that the induction of IN from the inside of the bacterium by inner membrane anchored INPs facing the luminal inner membrane side is very similar to IN induced by bacterial INPs located at the outer membrane. Bacterial ghosts derived from these different constructs showed first droplet freezing values between -6 and -8 °C, whereas E. coli C41 BGs alone without carrying inner membrane anchored INPs exhibit a T50 of -18.9 °C. Sum frequency generation spectroscopy showed structural ordered water at the BG/water interface, which increased close to the water melting point. Together, this indicates that the more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of inner membrane anchored INP constructs to ultrapure water filling the inner space of the BGs.

Halobacterium salinarum virus ChaoS9, a Novel Halovirus Related to PhiH1 and PhiCh1.

The unexpected lysis of a large culture of Halobacterium salinarum strain S9 was found to be caused by a novel myovirus, designated ChaoS9. Virus purification from the culture lysate revealed a homogeneous population of caudovirus-like particles. The viral genome is linear, dsDNA that is partially redundant and circularly permuted, has a unit length of 55,145 nt, a G + C% of 65.3, and has 85 predicted coding sequences (CDS) and one tRNA (Arg) gene. The left arm of the genome (0⁻28 kbp) encodes proteins similar in sequence to those from known caudoviruses and was most similar to myohaloviruses phiCh1 (host: Natrialbamagadii) and phiH1 (host: Hbt. salinarum). It carries a tail-fiber gene module similar to the invertible modules present in phiH1 and phiCh1. However, while the tail genes of ChaoS9 were similar to those of phiCh1 and phiH1, the Mcp of ChaoS9 was most similar (36% aa identity) to that of Haloarcula hispanica tailed virus 1 (HHTV-1). Provirus elements related to ChaoS9 showed most similarity to tail/assembly proteins but varied in their similarity with head/assembly proteins. The right arm (29⁻55 kbp) of ChaoS9 encoded proteins involved in DNA replication (ParA, RepH, and Orc1) but the other proteins showed little similarity to those from phiH1, phiCh1, or provirus elements, and most of them could not be assigned a function. ChaoS9 is probably best classified within the genus Myohalovirus, as it shares many characteristics with phiH1 (and phiCh1), including many similar proteins. However, the head/assembly gene region appears to have undergone a recombination event, and the inferred proteins are different to those of phiH1 and phiCh1, including the major capsid protein. This makes the taxonomic classification of ChaoS9 more ambiguous. We also report a revised genome sequence and annotation of Natrialba virus phiCh1.

Complete Genome Sequence of the Model Halovirus PhiH1 (ΦH1).

The halophilic myohalovirus Halobacterium virus phiH (ΦH) was first described in 1982 and was isolated from a spontaneously lysed culture of Halobacterium salinarum strain R1. Until 1994, it was used extensively as a model to study the molecular genetics of haloarchaea, but only parts of the viral genome were sequenced during this period. Using Sanger sequencing combined with high-coverage Illumina sequencing, the full genome sequence of the major variant (phiH1) of this halovirus has been determined. The dsDNA genome is 58,072 bp in length and carries 97 protein-coding genes. We have integrated this information with the previously described transcription mapping data. PhiH could be classified into Myoviridae Type1, Cluster 4 based on capsid assembly and structural proteins (VIRFAM). The closest relative was Natrialba virus phiCh1 (φCh1), which shared 63% nucleotide identity and displayed a high level of gene synteny. This close relationship was supported by phylogenetic tree reconstructions. The complete sequence of this historically important virus will allow its inclusion in studies of comparative genomics and virus diversity.

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.

In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadiiIMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

Functional display of ice nucleation protein InaZ on the surface of bacterial ghosts.

In a concept study the ability to induce heterogeneous ice formation by Bacterial Ghosts (BGs) from Escherichia coli carrying ice nucleation protein InaZ from Pseudomonas syringae in their outer membrane was investigated by a droplet-freezing assay of ultra-pure water. As determined by the median freezing temperature and cumulative ice nucleation spectra it could be demonstrated that both the living recombinant E. coli and their corresponding BGs functionally display InaZ on their surface. Under the production conditions chosen both samples belong to type II ice-nucleation particles inducing ice formation at a temperature range of between -5.6 °C and -6.7 °C, respectively. One advantage for the application of such BGs over their living recombinant mother bacteria is that they are non-living native cell envelopes retaining the biophysical properties of ice nucleation and do no longer represent genetically modified organisms (GMOs).

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Genomic manipulations in alkaliphilic haloarchaea demonstrated by a gene disruption in Natrialba magadii.

Alkaliphilic haloarchaea, a distinct physiological group from the closely related neutrophilic haloarchaea, represent an underutilized resource for basic research and industrial applications. In contrast to the neutrophilic haloarchaea, no reports on genomic manipulations in haloalkaliphiles have been published until now. Genomic manipulations via homologous recombination are useful for basic research. In this study, we demonstrate the possibility for this strategy in alkaliphilic haloarchaea for the first time. In a previous study, we developed a PEG-mediated transformation technique for alkaliphilic haloarchaea that was deployed in this study to deliver a gene disruption cassette into the model organism Natrialba magadii. The gene encoding for the well-studied Natrialba extracellular protease was successfully disrupted by a recombination marker gene, demonstrating a proof of principle for the usability of homologous recombination for genomic manipulations in alkaliphilic haloarchaea. Since halo(alkali)philic Archaea are polyploid, a selection process was applied in order to obtain a mutant strain containing exclusively disrupted genes. The resulting strain exhibited no proteolytic activity measurable by an azo-casein assay. Complementation was able to restore proteolytic activity. The expression pattern of the Natrialba extracellular protease was different in the complemented strain.

Genotyping of Burkholderia mallei from an outbreak of glanders in Bahrain suggests multiple introduction events.

BACKGROUND: Glanders, caused by the gram-negative bacterium Burkholderia mallei, is a highly infectious zoonotic disease of solipeds causing severe disease in animals and men. Although eradicated from many Western countries, it recently emerged in Asia, the Middle-East, Africa, and South America. Due to its rareness, little is known about outbreak dynamics of the disease and its epidemiology. METHODOLOGY/PRINCIPAL FINDINGS: We investigated a recent outbreak of glanders in Bahrain by applying high resolution genotyping (multiple locus variable number of tandem repeats, MLVA) and comparative whole genome sequencing to B. mallei isolated from infected horses and a camel. These results were compared to samples obtained from an outbreak in the United Arab Emirates in 2004, and further placed into a broader phylogeographic context based on previously published B. mallei data. The samples from the outbreak in Bahrain separated into two distinct clusters, suggesting a complex epidemiological background and evidence for the involvement of multiple B. mallei strains. Additionally, the samples from Bahrain were more closely related to B. mallei isolated from horses in the United Arab Emirates in 2004 than other B. mallei which is suggestive of repeated importation to the region from similar geographic sources. CONCLUSION/SIGNIFICANCE: High-resolution genotyping and comparative whole genome analysis revealed the same phylogenetic patterns among our samples. The close relationship of the Dubai/UAE B. mallei populations to each other may be indicative of a similar geographic origin that has yet to be identified for the infecting strains. The recent emergence of glanders in combination with worldwide horse trading might pose a new risk for human infections.

Antimicrobial activity of a chimeric enzybiotic towards Staphylococcus aureus.

Phage lytic enzymes (enzybiotics) have gained attention as prospective tools to eradicate Gram-positive pathogens resistant to antibiotics. Attempts to purify the P16 endolysin of Staphylococcus aureus phage P68 were unsuccessful owing to the poor solubility of the protein. To overcome this limitation, we constructed a chimeric endolysin (P16-17) comprised of the inferred N-terminal d-alanyl-glycyl endopeptidase domain and the C-terminal cell wall targeting domain of the S. aureus phage P16 endolysin and the P17 minor coat protein, respectively. The domain swapping approach and the applied purification procedure resulted in soluble P16-17 protein, which exhibited antimicrobial activity towards S. aureus. In addition, P16-17 augmented the antimicrobial efficacy of the antibiotic gentamicin. This synergistic effect could be useful to reduce the effective dose of aminoglycoside antibiotics.

Genetic diversity and phylogenetic relationships of bacteria belonging to the Ochrobactrum-Brucella group by recA and 16S rRNA gene-based comparative sequence analysis.

The genetic diversity and phylogenetic interrelationships among 106 Ochrobactrum strains (O. anthropi: 72, O. intermedium: 22, O. tritici: 5, O. oryzae: 2, O. grignonense: 2, O. gallinifaecis: 1, O. lupini: 2), the type strains of the eight Brucella species and other closely related taxa were studied by recA and rrs gene (16S rRNA) comparative sequence analysis. Both markers correctly delineated the various Ochrobactrum species; however, resolution at the subspecies level was considerably higher in the recA gene-based approach. Phylogenetic analyses using neighbor-joining, parsimony, and maximum likelihood algorithms generated trees with similar topologies but the overall branching order, and also the order of the subclades, were not stable in either assay, which could be explained by generally high recA and rrs sequence similarities. Ochrobactrum and Pseudochrobactrum formed separate clades distinct from other Alphaproteobacteria with Bartonella, Agrobacterium, and Rhizobium as the closest relatives. O. gallinifaecis was the most distinct member, when compared to the type species O. anthropi, with rrs and recA similarities of 96.2% and 81.4%. Brucella species were indistinguishable, exhibiting high rrs and recA gene similarities of 98.6% and 85.5% compared with Ochrobactrum intermedium. At the protein level, all RecA sequences among the various Ochrobactrum species and between Ochrobactrum and Brucella were highly similar with only a few amino acid substitutions. O. anthropi and O. tritici were indistinguishable by means of their RecA proteins. A set of initially biochemically classified strains did not cluster within their assigned species and they either grouped within other known species or grouped as potential novel Ochrobactrum species. In further investigations, these strains were reclassified and described as novel species. In summary, Ochrobactrum is a highly diverse genus comprising several novel species. We recommend recA- in addition to rrs gene-analysis for correct species allocation and subtyping of novel Ochrobactrum isolates.

Specific detection and differentiation of Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. by a multi-primer PCR that targets the recA gene.

Ochrobactrum anthropi, Ochrobactrum intermedium and Brucella spp. are phenotypically and genetically closely related pathogens that may cause disease with similar clinical presentation. Consequently, difficulties in their identification and differentiation have been reported. In this study, a sensitive recA gene-based multi-primer single-target PCR (MP-ST-PCR) was developed that allowed the specific detection and differentiation of these clinically relevant pathogens. The specificity of the assay was evaluated using a representative panel of 50 O. anthropi and 16 O. intermedium strains and the type strains of all Brucella spp. Detection limits for purified DNA from O. anthropi, O. intermedium and Brucella melitensis were 100, 10 and 100 fg, respectively. Brucella DNA was also successfully detected in various clinical specimens from a human patient with culture-proven brucellosis and from a Brucella-infected sheep and its aborted fetuses. The sensitivity of the MP-ST-PCR was comparable to that of an evaluated in-house Brucella real-time PCR assay. The developed assay closes a diagnostic gap and provides a simple but robust tool for the sensitive detection and correct identification of O. anthropi, O. intermedium and Brucella spp.

Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis.

A recA-PCR restriction fragment length polymorphism assay was developed to study intraspecies variation among Ochrobactrum anthropi. Primers deduced from the known recA gene sequence of the genetically closely related genus Brucella allowed the specific amplification of a 1065 bp recA fragment from each of the 38 O. anthropi and the eight Brucella strains investigated. RecA was also amplified from the type strains of O. intermedium, O. tritici, and O. lupini but could not be generated from O. grignonense and O. gallinifaecis. Subsequent comparative recA sequence- and HaeIII-recA restriction fragment length polymorphism analysis identified nine different genospecies among the tested 38 O. anthropi isolates, whereas the recA sequences of the Brucella spp. were indistinguishable. Furthermore, Brucella spp., O. anthropi, O. intermedium, and O. tritici were clearly separated from each other by means of their recA sequences and HaeIII restriction patterns. Five strains of uncertain species status listed in the Culture Collection University of Göteborg bacterial culture collection as O. anthropi were characterized by recA analysis, and their phylogenetic position within the Brucella-Ochrobactrum group was determined. In summary, recA-sequence analysis provides a new reliable molecular subtyping tool to study the phylogeny of the Ochrobactrum taxon at both the inter- and intraspecies level.

Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay.

A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344(T). B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.

Detection of Chromobacterium violaceum by multiplex PCR targeting the prgI, spaO, invG, and sipB genes.

Based on the recently completed genomic sequence of Chromobacterium violaceum American Type Culture Collection (ATCC) 12472 a multiplex PCR assay targeting the prgI, spaO, invG, and sipB genes of the Salmonella SPI-1 homologue type-III secretion system was developed. PCR products of 255bp (prgI), 749bp (spaO), 1685bp (invG), and 1752bp (sipB) were successfully amplified simultaneously in a single reaction with all Chr. violaceum strains investigated whereas other bacteria tested negative. The detection limit for pure cultures in multiplex PCR analysis was 100CFU. The developed assay significantly improves rapid identification of Chr. violaceum and allows its differentiation from closely related organisms.

Functional analysis of the lysis genes of Staphylococcus aureus phage P68 in Escherichia coli.

Double-stranded DNA phages of both Gram-positive and Gram-negative bacteria typically use a holin-endolysin system to achieve lysis of their host. In this study, the lysis genes of Staphylococcus aureus phage P68 were characterized. P68 gene lys16 was shown to encode a cell-wall-degrading enzyme, which causes cell lysis when externally added to clinical isolates of S. aureus. Another gene, hol15, was identified embedded in the -1 reading frame at the 3' end of lys16. The deduced Hol15 protein has three putative transmembrane domains, and thus resembles class I holins. An additional candidate holin gene, hol12, was found downstream of the endolysin gene lys16 based on two predicted transmembrane domains of the encoded protein, which is a typical trait of class II holins. The synthesis of either Hol12 or Hol15 resulted in growth retardation of Escherichia coli, and both hol15 and hol12 were able to complement a phage lambda Sam mutation. The hol15 gene has a dual start motif beginning with the codons Met1-Lys2-Met3.... Evidence is presented that the hol15 gene encodes a lysis inhibitor (anti-holin) and a lysis effector (actual holin). As depolarization of the membrane converted the anti-holin to a functional holin, these studies suggested that hol15 functions as a typical dual start motif class I holin. The unusual arrangement of the P68 lysis genes is discussed.

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis.

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.

Complete nucleotide sequence and molecular characterization of two lytic Staphylococcus aureus phages: 44AHJD and P68.

The first complete nucleotide sequences of two lytic Staphylococcus aureus double stranded DNA phages, 44AHJD (16784 bp) and P68 (18227 bp), are reported. Both are small isometric phages, with short, non-contractile tails and a pre-neck appendage. Based on their morphology, their genome size, the similarity of the encoded gene products, the type of infection and on the possession of a type B DNA polymerase, 44AHJD and P68 are allocated to the order Caudovirales, family Podoviridae, genus 'phi29-like phages'. The genome of 44AHJD differs from that of P68 by a deletion spanning nucleotides 10091 to 11531 of the P68 genome. The electrophoretic analysis of the terminal DNA fragments of P68 DNA and P68 DNA protein complex suggested the presence of a terminal protein at either DNA end. In contrast to the lysis cassette of the phi29-like phages, which is located at the end of the late operon, the lysis cassette of 44AHJD and P68 is located within the structural genes.