Identification and onion pathogenicity of Burkholderia cepacia complex isolates from the onion rhizosphere and onion field soil

Burkholderia cepacia complex strains are genetically related but phenotypically diverse organisms that are important opportunistic pathogens in patients with cystic fibrosis (CF,) as well as pathogens of onion and banana, colonizers of the rhizospheres of many plant species, and common inhabitants of bulk soil. Genotypic identification and pathogenicity characterization were performed on B. cepacia complex isolates from the rhizosphere of onion and organic soils in Michigan. A total of 3,798 putative B. cepacia complex isolates were recovered on Pseudomonas cepacia azelaic acid tryptamine and trypan blue tetracycline semiselective media during the 2004 growing season from six commercial onion fields located in two counties in Michigan. Putative B. cepacia complex isolates were identified by hybridization to a 16S rRNA gene probe, followed by duplex PCR using primers targeted to the 16S rRNA gene and recA sequences and restriction fragment length polymorphism analysis of the recA sequence. A total of 1,290 isolates, 980 rhizosphere and 310 soil isolates, were assigned to the species B. cepacia (160), B. cenocepacia (480), B. ambifaria (623), and B. pyrrocinia (27). The majority of isolates identified as B. cepacia (85%), B. cenocepacia (90%), and B. ambifaria (76%) were pathogenic in a detached onion bulb scale assay and caused symptoms of water soaking, maceration, and/or necrosis. A phylogenetic analysis of recA sequences from representative B. cepacia complex type and panel strains, along with isolates collected in this study, revealed that the B. cenocepacia isolates associated with onion grouped within the III-B lineage and that some strains were closely related to strain AU1054, which was isolated from a CF patient. This study revealed that multiple B. cepacia complex species colonize the onion rhizosphere and have the potential to cause sour skin rot disease of onion. In addition, the onion rhizosphere is a natural habitat and a potential environmental source of B. cenocepacia.     

Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Use of suppression-subtractive hybridization to identify genes in the Burkholderia cepacia complex that are unique to Burkholderia cenocepacia

We have previously shown differences in virulence between species of the Burkholderia cepacia complex using the alfalfa infection model and the rat agar bead chronic infection model. Burkholderia cenocepacia strains were more virulent in these two infection models than Burkholderia multivorans and Burkholderia stabilis strains. In order to identify genes that may account for the increased virulence of B. cenocepacia, suppression-subtractive hybridization was performed between B. cenocepacia K56-2 and B. multivorans C5393 and between B. cenocepacia K56-2 and B. stabilis LMG14294. Genes identified included DNA modification/phage-related/insertion sequences and genes involved in cell membrane/surface structures, resistance, transport, metabolism, regulation, secretion systems, as well as genes of unknown function. Several of these genes were present in the ET12 lineage of B. cenocepacia but not in other members of the B. cepacia complex. Virulence studies in a chronic lung infection model determined that the hypothetical YfjI protein, which is unique to the ET12 clone, contributes to lung pathology. Other genes specific to B. cenocepacia and/or the ET12 lineage were shown to play a role in biofilm formation and swarming or swimming motility.     

Sequence divergence in type III secretion gene clusters of the Burkholderia cepacia complex

The Burkholderia cepacia complex (BCC) comprises a group of bacteria associated with opportunistic infections, especially in cystic fibrosis patients. B. cenocepacia J2315, of the transmissible ET12 lineage, contains a type III secretion (TTS) gene cluster implicated in pathogenicity. PCR and hybridisation assays indicate that the TTS gene cluster is present in all members of the BCC except B. cepacia (formerly genomovar I). The TTS gene clusters of B. cenocepacia J2315 and B. multivorans are similar in organisation but have variable levels of gene identity. Nucleotide sequence data obtained for the equivalent region of the B. cepacia genome indicate the absence of TTS structural genes due to a rearrangement likely to involve more than one step.     

The role of prophage-like elements in the diversity of Salmonella enterica serovars

The Salmonella enterica serovar Typhi CT18 (S.Typhi) chromosome harbours seven distinct prophage-like elements, some of which may encode functional bacteriophages. In silico analyses were used to investigate these regions in S.Typhi CT18, and ultimately compare these integrated bacteriophages against 40 other Salmonella isolates using DNA microarray technology. S.Typhi CT18 contains prophages that show similarity to the lambda, Mu, P2 and P4 bacteriophage families. When compared to other S.Typhi isolates, these elements were generally conserved, supporting a clonal origin of this serovar. However, distinct variation was detected within a broad range of Salmonella serovars; many of the prophage regions are predicted to be specific to S.Typhi. Some of the P2 family prophage analysed have the potential to carry non-essential "cargo" genes within the hyper-variable tail region, an observation that suggests that these bacteriophage may confer a level of specialisation on their host. Lysogenic bacteriophages therefore play a crucial role in the generation of genetic diversity within S.enterica.     

Conservation of a novel protein associated with an antibiotic efflux operon in Burkholderia cenocepacia

Burkholderia cenocepacia is a significant problem in individuals with cystic fibrosis and is a member of the B. cepacia complex of closely related antibiotic resistant bacteria. A salicylate-regulated antibiotic efflux operon has been identified in B. cenocepacia and one of its four genes, llpE, is without parallel in previously reported efflux operons. PCR amplification and sequencing of llpE from B. cepacia complex isolates demonstrated the highest prevalence in B. cenocepacia with a high degree of sequence conservation. While at least one non-synonymous mutation was identified between isolates from different genomovars, only synonymous differences were identified within the IIIA and IIIB sub-groups of B. cenocepacia. Structural modeling suggests that LlpE is a member of the alpha/beta hydrolase enzyme family. Identification of strong structural homology to hydrolases and a high degree of conservation in B. cenocepacia suggests an enzymatic function for LlpE, benefiting survival in the cystic fibrosis lung.     

Bacteriophage Mu genome sequence: analysis and comparison with Mu-like prophages in Haemophilus, Neisseria and Deinococcus

We report the complete 36,717 bp genome sequence of bacteriophage Mu and provide an analysis of the sequence, both with regard to the new genes and other genetic features revealed by the sequence itself and by a comparison to eight complete or nearly complete Mu-like prophage genomes found in the genomes of a diverse group of bacteria. The comparative studies confirm that members of the Mu-related family of phage genomes are genetically mosaic with respect to each other, as seen in other groups of phages such as the phage lambda-related group of phages of enteric hosts and the phage L5-related group of mycobacteriophages. Mu also possesses segments of similarity, typically gene-sized, to genomes of otherwise non-Mu-like phages. The comparisons show that some well-known features of the Mu genome, including the invertible segment encoding tail fiber sequences, are not present in most members of the Mu genome sequence family examined here, suggesting that their presence may be relatively volatile over evolutionary time.The head and tail-encoding structural genes of Mu have only very weak similarity to the corresponding genes of other well-studied phage types. However, these weak similarities, and in some cases biochemical data, can be used to establish tentative functional assignments for 12 of the head and tail genes. These assignments are strongly supported by the fact that the order of gene functions assigned in this way conforms to the strongly conserved order of head and tail genes established in a wide variety of other phages. We show that the Mu head assembly scaffolding protein is encoded by a gene nested in-frame within the C-terminal half of another gene that encodes the putative head maturation protease. This is reminiscent of the arrangement established for phage lambda.     

Incidence of the cblA major subunit pilin gene amongst Burkholderia species

PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.     

Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.     

Stable Non-Mutator Stocks of Maize Have Sequences Homologous to the Mu1 Transposable Element

Mutator stocks of maize produce mutants at many loci at rates 20- to 50-fold above spontaneous levels. Current evidence suggests that this high mutation rate is mediated by an active transposable element system, Mu. Members of this transposable element family are found in approximately 10-60 copies in Mutator stocks. We report here an initial characterization of previously undetected sequences homologous to Mu elements in eight non-Mutator inbred lines and varieties of maize that have a normal low mutation rate. All stocks have approximately 40 copies of sequences homologous only to the terminal repeat and show weak homology to an internal probe. In addition, several of the stocks contain an intact Mu element. One intact Mu element and two terminal-specific clones have been isolated from one non-Mutator line, B37. The cloned sequences have been used to demonstrate that in genomic DNA the intact element, termed Mu1.4B37, is modified, such that restriction sites in its termini are not accessible to cleavage by the HinfI restriction enzyme. This modification is similar to that observed in Mutator lines that have lost activity. We hypothesize that the DNA modification of the Mu-like element may contribute to the lack of Mutator activity in B37.     

Diverse pathogenicity of Burkholderia cepacia complex strains in the Caenorhabditis elegans host model

A fast screening method was developed to assess the pathogenicity of a diverse collection of environmental and clinical Burkholderia cepacia complex isolates in the nematode Caenorhabditis elegans. The method was validated by comparison with the standard slow-killing assay. We observed that the pathogenicity of B. cepacia complex isolates in C. elegans was strain-dependent but species-independent. The wide range of observed pathogenic phenotypes agrees with the high degree of phenotypic variation among species of the B. cepacia complex and suggests that the taxonomic classification of a given strain within the complex cannot predict pathogenicity.     

Suppression-subtractive hybridisation reveals variations in gene distribution amongst the<Emphasis Type="Italic"> Burkholderia cepacia</Emphasis> complex,including the presence in some strains of a genomic island containing putative polysaccharide production genes

Some strains of the Burkholderia cepacia complex, including the ET12 lineage, have been implicated in epidemic spread amongst cystic fibrosis (CF) patients. Suppression-subtractive hybridisation was used to identify genomic regions within strain J2315 (ET12 lineage; genomovar IIIA) that were absent from a non-transmissible genomovar IIIB strain. Sequence data from 15 subtracted clones were used to interrogate the genome sequence of strain J2315 and identify genomic regions incorporating the subtracted sequences. Many of the genomic regions displayed abnormally low GC content and similarity to sequences implicated in gene transfer. The distribution of three subtracted regions amongst members of the B. cepacia complex varied. A large cluster of genes with strong sequence similarity to capsular production genes from Burkholderia mallei and other bacterial pathogens was identified. This genomic island was detected in some but not all representatives of genomovar IIIA, two out of four genomovar I strains, and one of two strains of Burkholderia multivorans, but was not detected in Burkholderia stabilis, Burkholderia vietnamiensis, genomovar VI or Burkholderia. ambifaria. The polysaccharide production gene cluster of strain J2315 carries an IS 407-like sequence within the gene similar to B. mallei wcbO that is lacking in other ET12 isolates. Genes from this cluster are expressed during exponential growth in broth.     

Transposition of the heat-stable toxin astA gene into a gifsy-2-related prophage of Salmonella enterica serovar Abortusovis

The horizontal transfer and acquisition of virulence genes via mobile genetic elements have been a major driving force in the evolution of Salmonella pathogenicity. Serovars of Salmonella enterica carry variable assortments of phage-encoded virulence genes, suggesting that temperate phages play a pivotal role in this process. Epidemic isolates of S. enterica serovar Typhimurium are consistently lysogenic for two lambdoid phages, Gifsy-1 and Gifsy-2, carrying known virulence genes. Other serovars of S. enterica, including serovars Dublin, Gallinarum, Enteritidis, and Hadar, carry distinct prophages with similarity to the Gifsy phages. In this study, we analyzed Gifsy-related loci from S. enterica serovar Abortusovis, a pathogen associated exclusively with ovine infection. A cryptic prophage, closely related to serovar Typhimurium phage Gifsy-2, was identified. This element, named Gifsy-2AO, was shown to contribute to serovar Abortusovis systemic infection in lambs. Sequence analysis of the prophage b region showed a large deletion which covers genes encoding phage tail fiber proteins and putative virulence factors, including type III secreted effector protein SseI (GtgB, SrfH). This deletion was identified in most of the serovar Abortusovis isolates tested and might be dependent on the replicative transposition of an adjacent insertion sequence, IS1414, previously identified in pathogenic Escherichia coli strains. IS1414 encodes heat-stable toxin EAST1 (astA) and showed multiple genomic copies in isolates of serovar Abortusovis. To our knowledge, this is the first evidence of intergeneric transfer of virulence genes via insertion sequence elements in Salmonella. The acquisition of IS1414 (EAST1) and its frequent transposition within the chromosome might improve the fitness of serovar Abortusovis within its narrow ecological niche.     

Events following prophage Mu induction.

Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.     

Novel non-specific DNA adenine methyltransferases

The mom gene of bacteriophage Mu encodes an enzyme that converts adenine to N(6)-(1-acetamido)-adenine in the phage DNA and thereby protects the viral genome from cleavage by a wide variety of restriction endonucleases. Mu-like prophage sequences present in Haemophilus influenzae Rd (FluMu), Neisseria meningitidis type A strain Z2491 (Pnme1) and H. influenzae biotype aegyptius ATCC 11116 do not possess a Mom-encoding gene. Instead, at the position occupied by mom in Mu they carry an unrelated gene that encodes a protein with homology to DNA adenine N(6)-methyltransferases (hin1523, nma1821, hia5, respectively). Products of the hin1523, hia5 and nma1821 genes modify adenine residues to N(6)-methyladenine, both in vitro and in vivo. All of these enzymes catalyzed extensive DNA methylation; most notably the Hia5 protein caused the methylation of 61% of the adenines in λ DNA. Kinetic analysis of oligonucleotide methylation suggests that all adenine residues in DNA, with the possible exception of poly(A)-tracts, constitute substrates for the Hia5 and Hin1523 enzymes. Their potential 'sequence specificity' could be summarized as AB or BA (where B = C, G or T). Plasmid DNA isolated from Escherichia coli cells overexpressing these novel DNA methyltransferases was resistant to cleavage by many restriction enzymes sensitive to adenine methylation.     

DNA Rearrangements Associated with Reversion of Bacteriophage Mu-Induced Mutations

Excision of transposable genetic elements from host DNA is different from the classical prophage lambda type of excision in that it occurs at low frequency and is mostly imprecise; only a minority of excision events restores the wild-type host sequences. In bacteriophage Mu, a highly efficient transposon, imprecise excision is 10-100 times more frequent than precise excision. We have examined a large number of these excision events by starting with mucts X mutants located in the Z gene of the lac operon of Escherichia coli. Mucts X mutants are defective prophages whose excision occurs at a measurable frequency. Imprecise excision was monitored by selecting for melibiose+ (Mel+) phenotype, which requires only a functioning lacY gene. Mel+ revertants exhibit an array of DNA rearrangements and fall in four main classes, the predominant one being comprised of revertants that have no detectable Mu DNA. Most of these revertants can further revert to Lac+. Perhaps 5 base-pair duplications, originally present at prophage-host junctions, are left in these lacZ-Y+ revertants, and they can be further repaired to lacZ+. Another class has, in addition to the loss of Mu DNA, deletions that extend generally, but not always, to only one side of the prophage. The other two classes of revertants, surprisingly, still have Mu DNA in the lacZ gene. One class has deletions in the Z gene, whereas, no deletions can be detected in the other. Many of the revertants in the last class can further revert to lacZ+, indicating that the lacY gene must have been turned on by a rearrangement within Mu DNA. Apparently, all of the detectable precise and most of the imprecise excision events require functioning of the Mu A gene. We suggest that a block in large-scale Mu replication allows the excision process to proceed.     

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.