Genomes and characterization of phages Bcep22 and BcepIL02, founders of a novel phage type in Burkholderia cenocepacia.

Within the Burkholderia cepacia complex, B. cenocepacia is the most common species associated with aggressive infections in the lungs of cystic fibrosis patients, causing disease that is often refractive to treatment by antibiotics. Phage therapy may be a potential alternative form of treatment for these infections. Here we describe the genome of the previously described therapeutic B. cenocepacia podophage BcepIL02 and its close relative, Bcep22. Phage Bcep22 was found to contain a circularly permuted genome of 63,882 bp containing 77 genes; BcepIL02 was found to be 62,714 bp and contains 76 predicted genes. Major virion-associated proteins were identified by proteomic analysis. We propose that these phages comprise the founding members of a novel podophage lineage, the Bcep22-like phages. Among the interesting features of these phages are a series of tandemly repeated putative tail fiber genes that are similar to each other and also to one or more such genes in the other phages. Both phages also contain an extremely large (ca. 4,600-amino-acid), virion-associated, multidomain protein that accounts for over 20% of the phages' coding capacity, is widely distributed among other bacterial and phage genomes, and may be involved in facilitating DNA entry in both phage and other mobile DNA elements. The phages, which were previously presumed to be virulent, show evidence of a temperate lifestyle but are apparently unable to form stable lysogens in their hosts. This ambiguity complicates determination of a phage lifestyle, a key consideration in the selection of therapeutic phages.

Efficacy of bacteriophage therapy in a model of Burkholderia cenocepacia pulmonary infection.

The therapeutic potential of bacteriophages (phages) in a mouse model of acute Burkholderia cenocepacia pulmonary infection was assessed. Phage treatment was administered by either intranasal inhalation or intraperitoneal injection. Bacterial density, macrophage inflammatory protein 2 (MIP-2), and tumor necrosis factor alpha (TNF-alpha) levels were significantly reduced in lungs of mice treated with intraperitoneal phages (P < .05). No significant differences in lung bacterial density or MIP-2 levels were found between untreated mice and mice treated with intranasal phages, intraperitoneal ultraviolet-inactivated phages, or intraperitoneal lambda phage control mice. Mock-infected mice treated with phage showed no significant increase in lung MIP-2 or TNF-alpha levels compared with mock-infected/mock-treated mice. We have demonstrated the efficacy of phage therapy in an acute B. cenocepacia lung infection model. Systemic phage administration was more effective than inhalational administration, suggesting that circulating phages have better access to bacteria in lungs than do topical phages.

Regulation of a phage endolysin by disulfide caging.

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz(P1), of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz(103), the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz(103) does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz(103) is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz(P1) and Lyz(103) define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz(103) is the first enzyme found to be regulated by disulfide bond caging of its active site.

Genomic and biological analysis of phage Xfas53 and related prophages of Xylella fastidiosa.

We report the plaque propagation and genomic analysis of Xfas53, a temperate phage of Xylella fastidiosa. Xfas53 was isolated from supernatants of X. fastidiosa strain 53 and forms plaques on the sequenced strain Temecula. Xfas53 forms short-tailed virions, morphologically similar to podophage P22. The 36.7-kb genome is predicted to encode 45 proteins. The Xfas53 terminase and structural genes are related at a protein and gene order level to P22. The left arm of the Xfas53 genome has over 90% nucleotide identity to multiple prophage elements of the sequenced X. fastidiosa strains. This arm encodes proteins involved in DNA metabolism, integration, and lysogenic control. In contrast to Xfas53, each of these prophages encodes head and DNA packaging proteins related to the siphophage lambda and tail morphogenesis proteins related to those of myophage P2. Therefore, it appears that Xfas53 was formed by recombination between a widespread family of X. fastidiosa P2-related prophage elements and a podophage distantly related to phage P22. The lysis cassette of Xfas53 is predicted to encode a pinholin, a signal anchor and release (SAR) endolysin, and Rz and Rz1 equivalents. The holin gene encodes a pinholin and appears to be subject to an unprecedented degree of negative regulation at both the level of expression, with rho-independent transcriptional termination and RNA structure-dependent translational repression, and the level of holin function, with two upstream translational starts predicted to encode antiholin products. A notable feature of Xfas53 and related prophages is the presence of 220- to 390-nucleotide degenerate tandem direct repeats encoding putative DNA binding proteins. Additionally, each phage encodes at least two BroN domain-containing proteins possibly involved in lysogenic control. Xfas53 exhibits unusually slow adsorption kinetics, possibly an adaptation to the confined niche of its slow-growing host.

The final step in the phage infection cycle: the Rz and Rz1 lysis proteins link the inner and outer membranes.

Bacteriophage lambda has four adjacent genes -S, R, Rz and Rz1- dedicated to host cell lysis. While S, encoding the holin and antiholin, and R, encoding the endolysin, have been intensively studied, the products of Rz and Rz1 have not been characterized at either the structural or functional levels. Rz1 is an outer membrane lipoprotein and our results indicate that Rz is a type II signal anchor protein. Here we present evidence that an Rz-Rz1 complex that spans the periplasm carries out the final step in the process of host lysis. These results are discussed in terms of a model where endolysin-mediated degradation of the cell wall is a prerequisite for conformational changes in the Rz-Rz1 complex leading to the juxtaposition and fusion of the IM and OM. Fusion of the two membranes removes the last physical barrier to efficient release of progeny virions.

Classification of Myoviridae bacteriophages using protein sequence similarity.

BACKGROUND: We advocate unifying classical and genomic classification of bacteriophages by integration of proteomic data and physicochemical parameters. Our previous application of this approach to the entirely sequenced members of the Podoviridae fully supported the current phage classification of the International Committee on Taxonomy of Viruses (ICTV). It appears that horizontal gene transfer generally does not totally obliterate evolutionary relationships between phages. RESULTS: CoreGenes/CoreExtractor proteome comparison techniques applied to 102 Myoviridae suggest the establishment of three subfamilies (Peduovirinae, Teequatrovirinae, the Spounavirinae) and eight new independent genera (Bcep781, BcepMu, FelixO1, HAP1, Bzx1, PB1, phiCD119, and phiKZ-like viruses). The Peduovirinae subfamily, derived from the P2-related phages, is composed of two distinct genera: the "P2-like viruses", and the "HP1-like viruses". At present, the more complex Teequatrovirinae subfamily has two genera, the "T4-like" and "KVP40-like viruses". In the genus "T4-like viruses" proper, four groups sharing >70% proteins are distinguished: T4-type, 44RR-type, RB43-type, and RB49-type viruses. The Spounavirinae contain the "SPO1-"and "Twort-like viruses." CONCLUSION: The hierarchical clustering of these groupings provide biologically significant subdivisions, which are consistent with our previous analysis of the Podoviridae.

Preparation of a phage DNA fragment library for whole genome shotgun sequencing.

The most efficient method to determine the genomic sequence of a dsDNA phage is to use a whole genome shotgun approach (WGSA). Preparation of a library where each genomic fragment has an equal chance of being represented is critical to the success of the WGSA. For many phages, there are regions of the genome likely to be under-represented in the shotgun library, which results in more gaps in the shotgun assembly than predicted by the Poisson distribution. However, as phage genomes are relatively small, this increased number of gaps does not present an insurmountable impediment to using the WGSA. This chapter will focus on construction of a high-quality random library and sequence analysis of this library in a 96-well format. Techniques are described for the mechanical fragmentation of genomic DNA into 2 kb average size fragments, preparation of the fragmented DNA for shotgun cloning, and advice on the choice of cloning vector for library preparation. Protocols for deepwell block culture, plasmid isolation, and sequencing in 96-well format are given. The rationale for determining the total number of random clones from a library to sequence for a 50 and 150 kb genome is explained. The steps involved in going from hundreds of shotgun sequencing traces to generating contigs will be outlined as well as how to close gaps in the sequence by primer walking on phage DNA and PCR-generated templates. Finally, examples will be given of how biological information about the phage genomic termini can be derived by analysis of the organization of individual clones in the shotgun sequence assembly. Specific examples are given for the circularly permuted termini of pac type phages, the direct terminal repeats found in most T7-like phages, variable host DNA at either end as in the Mu-like phages, and the 5' and 3' overhanging ends of cos type phages. The end result of these steps is the entire DNA sequence of a novel phage, ready for gene prediction.

Role of phages in the pathogenesis of Burkholderia, or 'Where are the toxin genes in Burkholderia phages?'.

Most bacteria of the genus Burkholderia are soil- and rhizosphere-associated, and rhizosphere associated, noted for their metabolic plasticity in the utilization of a wide range of organic compounds as carbon sources. Many Burkholderia species are also opportunistic human and plant pathogens, and the distinction between environmental, plant, and human pathogens is not always clear. Burkholderia phages are not uncommon and multiple cryptic prophages are identifiable in the sequenced Burkholderia genomes. Phages have played a crucial role in the transmission of virulence factors among many important pathogens; however, the data do not yet support a significant correlation between phages and pathogenicity in the Burkholderia. This may be due to the role of Burkholderia as a 'versaphile' such that selection is occurring in several niches, including as a pathogen and in the context of environmental survival.

Rz/Rz1 lysis gene equivalents in phages of Gram-negative hosts.

Under usual laboratory conditions, lysis by bacteriophage lambda requires only the holin and endolysin genes, but not the Rz and Rz1 genes, of the lysis cassette. Defects in Rz or Rz1 block lysis only in the presence of high concentrations of divalent cations. The lambda Rz and Rz1 lysis genes are remarkable in that Rz1, encoding an outer membrane lipoprotein, is completely embedded in the +1 register within Rz, which itself encodes an integral inner membrane protein. While Rz and Rz1 equivalents have been identified in T7 and P2, most phages, including such well-studied classic phages as T4, P1, T1, Mu and SP6, lack annotated Rz/Rz1 equivalents. Here we report that a search strategy based primarily on gene arrangement and membrane localization signals rather than sequence similarity has revealed that Rz/Rz1 equivalents are nearly ubiquitous among phages of Gram-negative hosts, with 120 of 137 phages possessing genes that fit the search criteria. In the case of T4, a deletion of a non-overlapping gene pair pseT.2 and pseT.3 identified as Rz/Rz1 equivalents resulted in the same divalent cation-dependent lysis phenotype. Remarkably, in T1 and six other phages, Rz/Rz1 pairs were not found but a single gene encoding an outer membrane lipoprotein with a C-terminal transmembrane domain capable of integration into the inner membrane was identified. These proteins were named "spanins," since their protein products are predicted to span the periplasm providing a physical connection between the inner and outer membranes. The T1 spanin gene was shown to complement the lambda Rz-Rz1- lysis defect, indicating that spanins function as Rz/Rz1 equivalents. The widespread presence of Rz/Rz1 or their spanin equivalents in phages of Gram-negative hosts suggests a strong selective advantage and that their role in the ecology of these phages is greater than that inferred from the mild laboratory phenotype.

Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors.

We have isolated BcepMu, a Mu-like bacteriophage whose host range includes human pathogenic Burkholderia cenocepacia (formally B. cepacia genomovar III) isolates, and determined its complete 36748 bp genomic sequence. Like enteric bacteriophage Mu, the BcepMu genomic DNA is flanked by variable host sequences, a result of transposon-mediated replication. The BcepMu genome encodes 53 proteins, including capsid assembly components related to those of Mu, and tail sheath and tube proteins related to those of bacteriophage P2. Seventeen of the BcepMu genes were demonstrated to encode homotypic interacting domains by using a cI fusion system. Most BcepMu genes have close homologs to prophage elements present in the two published Salmonella typhi genomes, and in the database sequences of Photorhabdus luminescens, and Chromobacterium violaceum. These prophage elements, designated SalMu, PhotoMu and ChromoMu, respectively, are collinear with BcepMu through nearly their entire lengths and show only limited mosaicism, despite the divergent characters of their hosts. The BcepMu family of Mu-like phages has a number of notable differences from Mu. Most significantly, the critical left end region of BcepMu is inverted with respect to Mu, and the BcepMu family of transposases is clearly of a distinct lineage with different molecular requirements at the transposon ends. Interestingly, a survey of 33 B.cepacia complex strains indicated that the BcepMu prophage is widespread in human pathogenic B.cenocepacia ET12 lineage isolates, but not in isolates from the PHDC or Midwest lineages. Identified members of the BcepMu family all contain a gene possibly involved in bacterial pathogenicity, a homolog of the type-two-secretion component exeA, but only BcepMu also carries a lipopolysaccharide modification acyltransferase which may also contribute a pathogenicity factor.

Bacteriophages of wastewater foaming-associated filamentous Gordonia reduce host levels in raw activated sludge.

Filamentous bacteria are a normal and necessary component of the activated sludge wastewater treatment process, but the overgrowth of filamentous bacteria results in foaming and bulking associated disruptions. Bacteriophages, or phages, were investigated for their potential to reduce the titer of foaming bacteria in a mixed-microbial activated sludge matrix. Foaming-associated filamentous bacteria were isolated from activated sludge of a commercial wastewater treatment plan and identified as Gordonia species by 16S rDNA sequencing. Four representative phages were isolated that target G. malaquae and two un-named Gordonia species isolates. Electron microscopy revealed the phages to be siphophages with long tails. Three of the phages--GordTnk2, Gmala1, and GordDuk1--had very similar ~76 kb genomes, with >93% DNA identity. These genomes shared limited synteny with Rhodococcus equi phage ReqiDocB7 and Gordonia phage GTE7. In contrast, the genome of phage Gsput1 was smaller (43 kb) and was not similar enough to any known phage to be placed within an established phage type. Application of these four phages at MOIs of 5-15 significantly reduced Gordonia host levels in a wastewater sludge model by approximately 10-fold as compared to non-phage treated reactors. Phage control was observed for nine days after treatment.

Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillus fermentum.

BACKGROUND: Contamination of corn mash by lactic acid bacteria (LAB) reduces the efficiency of the ethanol fermentation process. The industry relies heavily on antibiotics for contamination control and there is a need to develop alternative methods. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses. RESULTS: Bacterial populations in 27 corn mash samples collected from nine different commercial plants were determined by pyrosequencing of 16S rRNA amplicons. The results showed that the most abundant bacteria (>50 % of total population) in 24 of the 27 samples included LAB genera such as Lactobacillus, Streptococcus, Lactococcus, Weissella, Enterococcus, and Pediococcus. Lactobacillus was identified as the most prevalent genus at all fermentation stages in all plants, accounting for between 2.3 and 93.7 % of each population and constituting the major genus (>50 %) in nine samples from five plants and the most abundant genus in five other samples. Lactobacillus species, including L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri were the most well-represented species. Two bacteriophages that target L. fermentum strains from ethanol plants, vB_LfeS_EcoSau and vB_LfeM_EcoInf (EcoSau and EcoInf), were isolated and characterized as a siphophage and a myophage, respectively. Analysis of the 31,703 bp genome of EcoSau revealed its similarity to the P335-like phage group, and the 106,701 bp genome of phage EcoInf was determined to be a novel phage type despite its distant relationship to the SPO1-like phages. Addition of phages EcoSau and EcoInf to L. fermentum-contaminated corn mash fermentation models restored the yields of ethanol and reduced levels of residual glucose, lactic acid, and acetic acid to that comparable to the infection-free control. CONCLUSIONS: This study provides detailed insight into the microbiota contaminating commercial ethanol fermentations, and highlights the abundance of LAB, especially L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri, in the process. This study suggests that phages with broad coverage of major LAB species can be applied directly to corn mash for antibiotic-free control of contamination in the ethanol fermentation industry.

Molecular characterization of the Calvin cycle enzyme phosphoribulokinase in the stramenopile alga Vaucheria litorea and the plastid hosting mollusc Elysia chlorotica.

Phosphoribulokinase (PRK), a nuclear-encoded plastid-localized enzyme unique to the photosynthetic carbon reduction (Calvin) cycle, was cloned and characterized from the stramenopile alga Vaucheria litorea. This alga is the source of plastids for the mollusc (sea slug) Elysia chlorotica which enable the animal to survive for months solely by photoautotrophic CO2 fixation. The 1633-bp V. litorea prk gene was cloned and the coding region, found to be interrupted by four introns, encodes a 405-amino acid protein. This protein contains the typical bipartite target sequence expected of nuclear-encoded proteins that are directed to complex (i.e. four membrane-bound) algal plastids. De novo synthesis of PRK and enzyme activity were detected in E. chlorotica in spite of having been starved of V. litorea for several months. Unlike the algal enzyme, PRK in the sea slug did not exhibit redox regulation. Two copies of partial PRK-encoding genes were isolated from both sea slug and aposymbiotic sea slug egg DNA using PCR. Each copy contains the nucleotide region spanning exon 1 and part of exon 2 of V. litorea prk, including the bipartite targeting peptide. However, the larger prk fragment also includes intron 1. The exon and intron sequences of prk in E. chlorotica and V. litorea are nearly identical. These data suggest that PRK is differentially regulated in V. litorea and E. chlorotica and at least a portion of the V. litorea nuclear PRK gene is present in sea slugs that have been starved for several months.

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex.

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.

Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses.

We have conducted a large-scale study of gene expression in the C4 monocot sorghum (Sorghum bicolor) L. Moench cv BTx623 in response to the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA), and the ethylene precursor aminocyclopropane carboxylic acid. Expression profiles were generated from seedling root and shoot tissue at 3 and 27 h, using a microarray containing 12,982 nonredundant elements. Data from 102 slides and quantitative reverse transcription-PCR data on mRNA abundance from 171 genes were collected and analyzed and are here made publicly available. Numerous gene clusters were identified in which expression was correlated with particular signaling compound and tissue combinations. Many genes previously implicated in defense responded to the treatments, including numerous pathogenesis-related genes and most members of the phenylpropanoid pathway, and several other genes that may represent novel activities or pathways. Genes of the octadecanoic acid pathway of jasmonic acid (JA) synthesis were induced by SA as well as by MeJA. The resulting hypothesis that increased SA could lead to increased endogenous JA production was confirmed by measurement of JA content. Comparison of responses to SA, MeJA, and combined SA+MeJA revealed patterns of one-way and mutual antagonisms, as well as synergistic effects on regulation of some genes. These experiments thus help further define the transcriptional results of cross talk between the SA and JA pathways and suggest that a subset of genes coregulated by SA and JA may comprise a uniquely evolved sector of plant signaling responsive cascades.