Novel Virulent and Broad-Host-Range Erwinia amylovora Bacteriophages Reveal a High Degree of Mosaicism and a Relationship to Enterobacteriaceae Phages

A diverse set of 24 novel phages infecting the fire blight pathogen Erwinia amylovora was isolated from fruit production environments in Switzerland. Based on initial screening, four phages (L1, M7, S6, and Y2) with broad host ranges were selected for detailed characterization and genome sequencing. Phage L1 is a member of the Podoviridae, with a 39.3-kbp genome featuring invariable genome ends with direct terminal repeats. Phage S6, another podovirus, was also found to possess direct terminal repeats but has a larger genome (74.7 kbp), and the virus particle exhibits a complex tail fiber structure. Phages M7 and Y2 both belong to the Myoviridae family and feature long, contractile tails and genomes of 84.7 kbp (M7) and 56.6 kbp (Y2), respectively, with direct terminal repeats. The architecture of all four phage genomes is typical for tailed phages, i.e., organized into function-specific gene clusters. All four phages completely lack genes or functions associated with lysogeny control, which correlates well with their broad host ranges and indicates strictly lytic (virulent) lifestyles without the possibility for host lysogenization. Comparative genomics revealed that M7 is similar to E. amylovora virus ΦEa21-4, whereas L1, S6, and Y2 are unrelated to any other E. amylovora phage. Instead, they feature similarities to enterobacterial viruses T7, N4, and ΦEcoM-GJ1. In a series of laboratory experiments, we provide proof of concept that specific two-phage cocktails offer the potential for biocontrol of the pathogen.     

Characterization of Two Virulent Phages of Lactobacillus plantarum

We characterized two Lactobacillus plantarum virulent siphophages, ATCC 8014-B1 (B1) and ATCC 8014-B2 (B2), previously isolated from corn silage and anaerobic sewage sludge, respectively. Phage B2 infected two of the eight L. plantarum strains tested, while phage B1 infected three. Phage adsorption was highly variable depending on the strain used. Phage defense systems were found in at least two L. plantarum strains, LMG9211 and WCSF1. The linear double-stranded DNA genome of the pac-type phage B1 had 38,002 bp, a G+C content of 47.6%, and 60 open reading frames (ORFs). Surprisingly, the phage B1 genome has 97% identity with that of Pediococcus damnosus phage clP1 and 77% identity with that of L. plantarum phage JL-1; these phages were isolated from sewage and cucumber fermentation, respectively. The double-stranded DNA (dsDNA) genome of the cos-type phage B2 had 80,618 bp, a G+C content of 36.9%, and 127 ORFs with similarities to those of Bacillus and Lactobacillus strains as well as phages. Some phage B2 genes were similar to ORFs from L. plantarum phage LP65 of the Myoviridae family. Additionally, 6 tRNAs were found in the phage B2 genome. Protein analysis revealed 13 (phage B1) and 9 (phage B2) structural proteins. To our knowledge, this is the first report describing such high identity between phage genomes infecting different genera of lactic acid bacteria.     

Characterization of Phages Virulent for Sarothamnus scoparius Bradyrhizobia

Four virulent phages—ΦDl, ΦTl, ΦCYT21, and ΦOS6, infective on Sarothamnus scoparius rhizobia—were isolated from the soil and characterized for morphology, host range, rate of adsorption to bacterial cells, and genome size. New phages were separated into two morphological families: Siphoviridae with long, noncontractile tails (ΦDl, ΦTl) and Myoviridae with long, contractile tails (ΦCYT21, ΦOS6). They were also classified into two groups by a host specificity. One of them included viruses (ΦDl and ΦTl) that lysed S. scoparius bradyrhizobia and Bradyrhizobium sp. (Lupinus) strain Dl, and the second one comprised phages (ΦCYT21 and ΦOS6) that parasitized only Scotch broom native microsymbionts. Phages specific for S. scoparius rhizobia were differentiated not only by morphology and host range but also by a genome size that was in the range from 47,583 to 60,098 b.p. An erratum to this article is available at .     

Xylella and Xanthomonas Mobil'omics

The gamma-proteobacterium Xanthomonadales groups two closely related genera of plant pathogens, Xanthomonas and Xylella. Whole genome sequencing and comparative analyses disclosed a high degree of identity and co-linearity of the chromosome backbone between species and strains. Differences observed are usually clustered into genomic islands, most of which are delimited by genetic mobile elements. Focus is given in this paper to describe which groups of mobile elements are found and what is the relative contribution of these elements to Xanthomonas and Xylella genomes. Insertion sequence (IS) elements have invaded the Xanthomonas genome several times, whereas Xylella is rich in phage-related regions. Also, different plasmids are found inhabiting the bacterial cells studied here. Altogether, these results suggest that the integrative elements such as phages and transposable elements as well as the episomal plasmids are important drivers of the genome evolution of this important group of plant pathogens.     

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU

Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.     

Mapping analysis of the Xylella fastidiosa genome

A cosmid library was made of the 2.7 Mb genome of the Gram-negative plant pathogenic bacterium Xylella fastidiosa and analysed by hybridisation mapping. Clones taken from the library as well as genomic restriction fragments of rarely cutting enzymes were used as probes. The latter served as a backbone for ordering the initial map contigs and thus facilitated gap closure. Also, the co-linearity of the cosmid map, and thus the eventual sequence, could be confirmed by this process. A subset of the eventual clone coverage was distributed to the Brazilian X.fastidiosa sequencing network. Data from this effort confirmed more quantitatively initial results from the hybridisation mapping that the redundancy of clone coverage ranged between 0 and 45-fold across the genome, while the average was 15-fold by experimental design. Reasons for this not unexpected fluctuation and the actual gaps are being discussed, as is the use of this effect for functional studies.     

Characterization of Regulatory Pathways in Xylella fastidiosa: Genes and Phenotypes Controlled by algU

Many virulence genes in plant bacterial pathogens are coordinately regulated by “global” regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.     

The Usage of Codons Which are Similar to Stop Codons in the Genomes of Xylella fastidiosa and Xanthomonas citri

During the evolution of living organisms, a natural selection event occurs toward the optimization of their genomes regarding the usage of codons. During this process which is known as codon bias, a set of preferred codons is naturally defined in the genome of a given organism, since there are 61 possible codons (plus 3 stop codons) to 20 amino acids. Such event leads to optimization of metabolic cellular processes such as translational efficiency, RNA stability and energy saving. Although we know why, we do not know how exactly a set of preferred codons for each amino acid is defined for a given genome considering that the usage frequency of each synonymous codons is peculiar to each organism. In order to help answering this question, we analyzed the usage frequency of codons which are similar to stop codons, since a minor mutation on these codons may lead to a stop codon into the open reading frame compromising the protein expression as a result. We found a reduced use of those codons in Xanthomomas axonopodis pv. citri which presents an optimized genome regarding codon usage. On the other hand, such codons are more often used in Xylella fastidiosa, which does not seem to have established codon preferences as previously shown. Our results support that a set of preferred codons is not randomly selected and propose new ideas to the field warranting further experiments in this regard.     

Genetic Analysis of a Novel Xylella fastidiosa Subspecies Found in the Southwestern United States

Xylella fastidiosa, the causal agent of several scorch diseases, is associated with leaf scorch symptoms in Chitalpa tashkentensis, a common ornamental landscape plant used throughout the southwestern United States. For a number of years, many chitalpa trees in southern New Mexico and Arizona exhibited leaf scorch symptoms, and the results from a regional survey show that chitalpa trees from New Mexico, Arizona, and California are frequently infected with X. fastidiosa. Phylogenetic analysis of multiple loci was used to compare the X. fastidiosa infecting chitalpa strains from New Mexico, Arizona, and trees imported into New Mexico nurseries with previously reported X. fastidiosa strains. Loci analyzed included the 16S ribosome, 16S-23S ribosomal intergenic spacer region, gyrase-B, simple sequence repeat sequences, X. fastidiosa-specific sequences, and the virulence-associated protein (VapD). This analysis indicates that the X. fastidiosa isolates associated with infected chitalpa trees in the Southwest are a highly related group that is distinct from the four previously defined taxons X. fastidiosa subsp. fastidiosa (piercei), X. fastidiosa subsp. multiplex, X. fastidiosa subsp. sandyi, and X. fastidiosa subsp. pauca. Therefore, the classification proposed for this new subspecies is X. fastidiosa subsp. tashke.Xylella fastidiosa is a gram-negative bacterium that multiplies within the xylem and causes serious disease problems in many diverse plant species. X. fastidiosa is considered a “new world” pathogen and is mainly found within North, Central, and South America (30). In many native plant species this bacterium exists as an apparently benign endophyte, while in other instances proliferation of X. fastidiosa within the xylem leads to disease typified by symptoms, including leaf scorch, chlorosis, stunting, branch dieback, inedible fruit, and eventually the death of the plant (4, 15). X. fastidiosa is transmitted by xylem-feeding insect vectors such as sharpshooters, leafhoppers, and spittle bugs (35). Diseases caused by X. fastidiosa include Pierce''s disease in grapes (7), citrus variegated chlorosis (CVC) (6), coffee leaf scorch (18), pecan leaf scorch (36), phony peach (41), plum leaf scald (32), and almond leaf scorch (25). X. fastidiosa has also been shown to be the causative agent of diseases found in landscape plants such as oleander leaf scorch (31), mulberry leaf scorch (14), and oak leaf scorch (3). In addition to the examples above proven through the completion of Koch''s postulates, X. fastidiosa is known to be associated with leaf scorch type diseases in several other ornamental landscape species including crape myrtle, olive, day lily, and southern magnolia (12).Chitalpa (Chitalpa tashkentensis Elias and Wisura) is an ornamental landscape plant that was developed for arid landscapes such as California, Arizona, Texas, and New Mexico. Chitalpa, originally bred in Russia and introduced into the United States in 1977, is an intergenic hybrid between desert willow (Chilopsis linearis Cav.) and Catalpa bignonioides Walt. (28). In the past, chitalpa trees across the Southwest were observed to display leaf scorch symptoms of unknown origin. X. fastidiosa was detected in many chitalpa trees that displayed leaf scorch symptoms in southern New Mexico (34). The first known occurrence of Pierce''s disease in New Mexico was reported in 2007, and the strains of X. fastidiosa found in infected New Mexico grapes were very similar to those present in chitalpa trees from the same area (33). The common use of chitalpa as a landscape plant in the Southwest coupled with the recent discovery that it can harbor X. fastidiosa strains similar to those associated with Pierce''s disease in New Mexico prompted a survey of chitalpa trees across the Southwest. The results of this survey show that chitalpa trees from New Mexico and Arizona are frequently infected with X. fastidiosa. Chitalpa plants imported into New Mexico nurseries from California were also found to contain similar strains of X. fastidiosa. A multilocus phylogenetic analysis was performed to further characterize these strains of X. fastidiosa. This analysis revealed that the X. fastidiosa isolates infecting chitalpa plants in New Mexico, Arizona, and imported into nurseries from California are highly related to each other and are distinct from the previously described subspecies fastidiosa (38).     

Prediction of potential thermostable proteins in Xylella fastidiosa

The average protein (E+K)/(Q+H) ratio in organisms has already been demonstrated to have a strong correlation with their optimal growth temperature. Employing the Thermo-Search web tool, we used this ratio as a basis to look for thermostable proteins in a mesophile, Xylella fastidiosa. Nine proteins were chosen to have their three-dimensional structures modeled by homology, using mainly proteins from mesophiles as templates. Resulting models featured a high number of hydrophobic interactions, a property that has been previously associated with thermostability. These results demonstrate the interesting possibility of using the (E+K)/(Q+H) ratio to find individual thermostable proteins in mesophilic organisms.     

Initial Genetic Analysis of Xylella fastidiosa in Texas

Xylella fastidiosa is the causative agent of Pierce’s Disease of grape. No published record of X. fastidiosa genetics in Texas exists despite growing financial risk to the U.S. grape industry, a Texas population of the glassy-winged sharpshooter insect vector (Homalodisca vitripennis) now spreading in California, and evidence that the bacterium is ubiquitous to southern states. Using sequences of conserved gyrB and mopB genes, we have established at least two strains in Texas, grape strain and ragweed strain, corresponding genetically with subsp. piercei and multiplex, respectively. The grape strain in Texas is found in Vitis vinifera varieties, hybrid vines, and wild Vitis near vineyards, whereas the ragweed strain in Texas is found in annuals, shrubs, and trees near vineyards or other areas. RFLP and QRT PCR techniques were used to differentiate grape and ragweed strains with greater efficiency than sequencing and are practical for screening numerous X. fastidiosa isolates for clade identity.     

Specific Detection of Xylella fastidiosa Pierce's Disease Strains

Pierce's disease (PD, Xylella fastidiosa) of grapevine is the primary pathogen limiting vinifera grape production in Florida and other regions of the southeastern United States. Quick and accurate detection of PD strains is essential for PD studies and control. A unique random amplified polymorphic DNA (PD1-1-2) was isolated from a PD strain from Florida. Fragment PD1-1-2 was cloned, sequenced, and found to be 1005 bp in length. PCR primers were designed to utilize these sequence data for PD strain detection. One primer set (XF176f–XF954r) amplified a 779-bp DNA fragment from 34 PD strains including seven pathotypes of X. fastidiosa, but not from strains of Xanthomonas campestris pv. campestris, Xan. vesicatoria or Escherichia coli. A second primer set (XF176f and XF686r) amplified a 511-bp fragment specific to 98 PD strains, but not from strains of citrus variegated chlorosis, mulberry leaf scorch, oak leaf scorch, periwinkle wilt, phony peach, or plum leaf scald. Sequence analysis indicated that RAPD fragment PD1-1-2 contains a Ser-tRNA gene. The PD-specific region includes a TaqI restriction site (TCGA) and is 150 bp downstream of the Ser-tRNA gene. Received: 1 March 1999 / Accepted: 5 April 1999     

Growth and siderophore production of Xylella fastidiosa under iron-limited conditions

In this study, the production of siderophores by Xylella fastidiosa from the citrus bacteria isolate 31b9a5c (FAPESP – ONSA, Brazil) was investigated. The preliminary evidence supporting the existence of siderophore in X. fastidiosa was found during the evaluation of sequencing data generated in our lab using the BLAST-X tool, which indicated putative open reading frames (ORFs) associated with iron-binding proteins. In an iron-limited medium siderophores were detected in the supernatant of X. fastidiosa cultures. The endophytic bacterium Methylobacterium extorquens was also evaluated. Capillary electrophoresis was used to separate putative siderophores produced by X. fastidiosa. The bacterial culture supernatants of X. fastidiosa were identified negative for hydroxamate and catechol and positive for M. extorquens that secreted hydroxamate-type siderophores.     

Surface motility of Xylella fastidiosa visualized by oblique illumination

Stereomicroscopic observations using oblique illuminations revealed the presence of two types of movement trails by Xylella fastidiosa strains (A- and G-genotypes) isolated from almond-leaf scorch samples on the surface of PW and PD3 culture media. The A-genotype strains showed curved motility trails, and the G-genotype strains showed straight motility trails. Haloes were found around some G-genotype colonies due to the excretion of unknown factors and (or) compounds, which might be related to bacterial surface motility.     

Genome Organization and Characterization of the Virulent Lactococcal Phage 1358 and Its Similarities to Listeria Phages

Virulent phage 1358 is the reference member of a rare group of phages infecting Lactococcus lactis. Electron microscopy revealed a typical icosahedral capsid connected to one of the smallest noncontractile tails found in a lactococcal phage of the Siphoviridae family. Microbiological characterization identified a burst size of 72 virions released per infected host cell and a latent period of 90 min. The host range of phage 1358 was limited to 3 out of the 60 lactococcal strains tested. Moreover, this phage was insensitive to four Abi systems (AbiK, AbiQ, AbiT, and AbiV). The genome of phage 1358 consisted of a linear, double-stranded, 36,892-bp DNA molecule containing 43 open reading frames (ORFs). At least 14 ORFs coded for structural proteins, as identified by SDS-PAGE coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. The genomic organization was similar to those of other siphophages. All genes were on the same coding strand and in the same orientation. This lactococcal phage was unique, however, in its 51.4% GC content, much higher than those of other phages infecting this low-GC Gram-positive host. A bias for GC-rich codons was also observed. Comparative analyses showed that several phage 1358 structural proteins shared similarity with two Listeria monocytogenes phages, P35 and P40. The possible origin and evolution of lactococcal phage 1358 is discussed.The first sequenced genome of a phage infecting Lactococcus lactis (bIL67) was reported in 1994 (57). Its genomic characterization was performed with the prospect of a better understanding of lactococcal phage biology. L. lactis is a Gram-positive bacterium added to milk to produce an array of fermented dairy products. In this human-made environment, substantial amounts of lactococcal cells are cultivated on a daily basis in large fermentation vats, and these added cells randomly encounter virulent phages present in heat-treated but nonsterile milk. Moreover, it is widely acknowledged that the increased use of the same bacterial strains within existing dairy facilities inevitably leads to milk fermentation failures due to the multiplication of virulent phages. This biotechnological problem reduces yields and lowers the quality of fermented products (51).Over 700 lactococcal phage isolates have been reported in the literature (3). To date, more than 25 complete genome sequences of lactococcal phages are publicly available in the NCBI database, and the sequencing of others is under way. These numbers indicate that Lactococcus phages are among the most studied of the bacterial viruses. All lactococcal phages belong to the order Caudovirales and are included within two families according to their tail morphology: the Siphoviridae (long noncontractile tail [most lactococcal phages]) and the Podoviridae (short noncontractile tail [few lactococcal phages]) (14). Currently, phages infecting L. lactis strains have been divided into 10 genetically distinct groups (14). The complete genomic sequence is available for at least one representative of 8 of the groups.Early sequencing efforts concentrated on the genomes of lactococcal phages belonging to the 936, c2, and P335 groups (Siphoviridae), because members of these groups were regularly isolated in dairy plants (8, 36, 50). PCR-based methods were also devised to rapidly classify these phages (41). These Siphoviridae phages pose a significant risk to the dairy industry, and their characterization is important for developing adapted antiphage strategies to limit their propagation and evolution.In recent years, representatives of the less recognized lactococcal phage groups have been characterized, including phages Q54 (22), KSY1 (13), 1706 (23), asccφ28 of the P034 group (39), and P087 (63). Their molecular characterizations were aimed at understanding why some phage groups (936, c2, and P335) predominate while the others have remained marginal, at best. However, it was recently reported that P034-like phages may be emerging in certain regions (52). Genomic and microbiological analyses indicated that members of these rare phage groups were likely the result of recombination between different lactococcal phages and phages infecting other Gram-positive bacteria, and they may not be fit to multiply rapidly in milk. For example, lactococcal phage 1706 shares similarities with Ruminococcus and Clostridium prophages (23). Similarly, L. lactis phage P087 structural proteins share identity with gene products found in a prophage in the Enterococcus faecalis genome (63). It was also shown previously that lactococcal phage asccφ28 was related to Streptococcus pneumoniae phage Cp-1 and Bacillus subtilis φ29-like phages (39). It was suggested that phages 1706, asccφ28, and P087 acquired a receptor-binding protein complex from another lactococcal phage that enabled them to infect a L. lactis host.Here, we report the complete genome sequence and analysis of phage 1358, a virulent representative of the 9th lactococcal phage group.     

Molecular models for shikimate pathway enzymes of Xylella fastidiosa

The Xylella fastidiosa is a bacterium that is the cause of citrus variegated chlorosis (CVC). The shikimate pathway is of pivotal importance for production of a plethora of aromatic compounds in plants, bacteria, and fungi. Putative structural differences in the enzymes from the shikimate pathway, between the proteins of bacterial origin and those of plants, could be used for the development of a drug for the control of CVC. However, inhibitors for shikimate pathway enzymes should have high specificity for X. fastidiosa enzymes, since they are also present in plants. In order to pave the way for structural and functional efforts towards antimicrobial agent development, here we describe the molecular modeling of seven enzymes of the shikimate pathway of X. fastidiosa. The structural models of shikimate pathway enzymes, complexed with inhibitors, strongly indicate that the previously identified inhibitors may also inhibit the X. fastidiosa enzymes.     

Strains of Xylella fastidiosa Rapidly Distinguished by Arbitrarily Primed-PCR

Genomic DNAs isolated from strains of Xylella fastidiosa that caused citrus variegated chlorosis, coffee leaf scorch, Pierce's Disease of grapevine, and plum leaf scorch were analyzed by arbitrarily primed polymerase chain reaction. Purified DNA was amplified under nonstringent conditions with single primers 21 nucleotides (nt) long. Thirty-nine amplification products were observed that were useful to distinguish among the strains and to derive a similarity matrix and construct a phenogram showing possible relationships among the strains. Strains isolated from diseased coffee and citrus in Brazil were closely related to each other (coefficient of similarity of 0.872), but only distantly related to a strain isolated from diseased grapevine in the USA (coefficient of similarity of 0.650). Strains of Xylella fastidiosa isolated from diseased plums in the USA and Brazil clustered with strains from different hosts isolated from their respective countries of origin. Thus, there may be two quite dissimilar clusters of strains of Xylella fastidiosa, one in North America and the other in South America. Each cluster contains strains that can cause disease in plum. The methods described provide a convenient and rapid method to distinguish between strains of Xylella fastidiosa that cause diseases of coffee and citrus in the same region of Brazil. This has not been possible previously. This will potentially enable the two strains to be distinguished in alternate hosts or in insect vectors. Received: 12 October 1999 / Accepted: 16 November 1999