Mark A. Schell

Type VI secretion is a major virulence determinant in Burkholderia mallei

Burkholderia mallei is a host‐adapted pathogen and a category B biothreat agent. Although the B. mallei VirAG two‐component regulatory system is required for virulence in hamsters, the virulence genes it regulates are unknown. Here we show with expression profiling that overexpression of virAG resulted in transcriptional activation of ∼60 genes, including some involved in capsule production, actin‐based intracellular motility, and type VI secretion (T6S). The 15 genes encoding the major sugar component of the homopolymeric capsule were up‐expressed > 2.5‐fold, but capsule was still produced in the absence of virAG. Actin tail formation required virAG as well as bimB, bimC and bimE, three previously uncharacterized genes that were activated four‐ to 15‐fold when VirAG was overproduced. Surprisingly, actin polymerization was found to be dispensable for virulence in hamsters. In contrast, genes encoding a T6S system were up‐expressed as much as 30‐fold and mutations in this T6S gene cluster resulted in strains that were avirulent in hamsters. SDS‐PAGE and mass spectrometry demonstrated that BMAA0742 was secreted by the T6S system when virAG was overexpressed. Purified His‐tagged BMAA0742 was recognized by glanders antiserum from a horse, a human and mice, indicating that this Hcp‐family protein is produced in vivo during infection.

Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is co‐ordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild‐type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3‐hydroxypalmitic acid methyl ester (3‐OH PAME) as the major component in the single peak of VEF activity. Authentic 3‐OH PAME and the purified VEF were active at ≤1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3‐OH PAME also increased the production of three virulence factors by a phcB mutant over 20‐fold to wild‐type levels, restored normal cell density‐associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small‐molecule S‐adenosylmethionine‐dependent methyltransferase, which might catalyse synthesis of 3‐OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3‐OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild‐type R. solanacearum. Other than acyl‐homoserine lactones, 3‐OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long‐distance intercellular communication.

Role of Extracellular Polysaccharide and Endoglucanase in Root Invasion and Colonization of Tomato Plants by Ralstonia solanacearum.

ABSTRACT Ralstonia solanacearum is a soilborne plant pathogen that normally invades hosts through their roots and then systemically colonizes aerial tissues. Previous research using wounded stem infection found that the major factor in causing wilt symptoms was the high-molecular-mass acidic extracellular polysaccharide (EPS I), but the beta-1,4-endoglucanase (EG) also contributes to virulence. We investigated the importance of EPS I and EG for invasion and colonization of tomato by infesting soil of 4-week-old potted plants with either a wild-type derivative or genetically well-defined mutants lacking EPS I, EG, or EPS I and EG. Bacteria of all strains were recovered from surface-disinfested roots and hypocotyls as soon as 4 h after inoculation; that bacteria were present internally was confirmed using immunofluorescence microscopy. However, the EPS-minus mutants did not colonize stems as rapidly as the wild type and the EG-minus mutant. Inoculations of wounded petioles also showed that, even though the mutants multiplied as well as the wild type in planta, EPS-minus strains did not spread as well throughout the plant stem. We conclude that poor colonization of stems by EPS-minus strains after petiole inoculation or soil infestation is due to reduced bacterial movement within plant stem tissues.

Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence

As reported previously for Ralstonia solanacearum strain GMI1000, wild‐type strains AW1 and K60 were shown to produce Hrp pili. AW1 and K60 mutants lacking Hrp pili still exhibited twitching motility, which requires type 4 pili (Tfp), and electron microscopy revealed that they still made flexuous polar pili. Twitching‐positive cells had an extracellular 17 kDa protein that was associated with piliation, and an internal 43‐amino‐acid sequence of this protein was typical of type 4 pilins. This amino acid sequence is encoded by an open reading frame, designated pilA, in the genomic sequence of GMI1000. PilA is 46% identical to a Pseudomonas aeruginosa type 4 pilin over its entire length and has all the conserved residues and motifs characteristic of type 4 group A pilins. pilA mutants did not make the 17 kDa PilA protein and did not exhibit twitching motility. When compared with its parent, an AW1 pilA mutant was reduced in virulence on tomato plants and in autoaggregation and biofilm formation in broth culture. Unlike AW1, a pilA mutant did not exhibit polar attachment to tobacco suspension culture cells or to tomato roots; it was also not naturally competent for transformation. We reported previously that twitching motility ceases in maturing AW1 colonies and that inactivation of PhcA, a global transcriptional regulator, results in colonies that continue to exhibit twitching motility. Similarly, in broth culture, expression of a pilA::lacZ fusion in AW1 decreased 10‐fold at high cell density, but expression remained high in a phcA mutant. In addition, pilA::lacZ expression was positively regulated 10‐fold by PehR, a response regulator that is known to be repressed by PhcA. This signal cascade is sufficient to explain why pilA expression, and thus twitching motility, decreases at high cell densities.

Bacterial genome adaptation to niches: Divergence of the potential virulence genes in three Burkholderia species of different survival strategies

BackgroundTwo closely related species Burkholderia mallei (Bm) and Burkholderia pseudomallei (Bp) are serious human health hazards and are potential bio-warfare agents, whereas another closely related species Burkholderia thailandensis (Bt) is a non-pathogenic saprophyte. To investigate the genomic factors resulting in such a dramatic difference, we first identified the Bm genes responsive to the mouse environment, and then examined the divergence of these genes in Bp and Bt.ResultsThe genes down-expressed, which largely encode cell growth-related proteins, are conserved well in all three species, whereas those up-expressed, which include potential virulence genes, are less well conserved or absent notably in Bt. However, a substantial number of up-expressed genes is still conserved in Bt. Bm and Bp further diverged from each other in a small number of genes resulting from unit number changes in simple sequence repeats (ssr) in the homologs.ConclusionOur data suggest that divergent evolution of a small set of genes, rather than acquisition or loss of pathogenic islands, is associated with the development of different life styles in these bacteria of similar genomic contents. Further divergence between Bm and Bp mediated by ssr changes may reflect different adaptive processes of Bm and Bp fine-tuning into their host environments.

Identification of Open Reading Frames Unique to a Select Agent: Ralstonia solanacearum Race 3 Biovar 2

An 8x draft genome was obtained and annotated for Ralstonia solanacearum race 3 biovar 2 (R3B2) strain UW551, a United States Department of Agriculture Select Agent isolated from geranium. The draft UW551 genome consisted of 80,169 reads resulting in 582 contigs containing 5,925,491 base pairs, with an average 64.5% GC content. Annotation revealed a predicted 4,454 protein coding open reading frames (ORFs), 43 tRNAs, and 5 rRNAs; 2,793 (or 62%) of the ORFs had a functional assignment. The UW551 genome was compared with the published genome of R. solanacearum race 1 biovar 3 tropical tomato strain GMI1000. The two phylogenetically distinct strains were at least 71% syntenic in gene organization. Most genes encoding known pathogenicity determinants, including predicted type III secreted effectors, appeared to be common to both strains. A total of 402 unique UW551 ORFs were identified, none of which had a best hit or >45% amino acid sequence identity with any R. solanacearum predicted protein; 16 had strong (E < 10(-13)) best hits to ORFs found in other bacterial plant pathogens. Many of the 402 unique genes were clustered, including 5 found in the hrp region and 38 contiguous, potential prophage genes. Conservation of some UW551 unique genes among R3B2 strains was examined by polymerase chain reaction among a group of 58 strains from different races and biovars, resulting in the identification of genes that may be potentially useful for diagnostic detection and identification of R3B2 strains. One 22-kb region that appears to be present in GMI1000 as a result of horizontal gene transfer is absent from UW551 and encodes enzymes that likely are essential for utilization of the three sugar alcohols that distinguish biovars 3 and 4 from biovars 1 and 2.

Transcriptional control of the nah and sal hydrocarbon-degradation operons by the nahR gene product

The positively regulated nah and sal operons of the NAH7 plasmid from Pseudomonas putida encode the enzymes for metabolism of naphthalene via salicylate. To study their coordinate regulation, a 6-kb DNA fragment containing the entire nahA gene (encoding naphthalene dioxygenase), the gene of the nah operon, was cloned into a RSF1010 plasmid derivative. Analysis of expression of nahA from the nah promoter in either Escherichia coli or Pseudomonas putida showed that a 1.6-kb DNA fragment from the nahR (nah operon regulatory locus) region was required in trans for (i) induction by salicylate; (ii) high-level expression of nahA, and (iii) complementation of nahR- mutants. Measurement of transcription in induced and uninduced P. putida showed that induction of the nah and sal operons occurred at the transcriptional level. The trans-acting positive regulatory gene, nahR, however, was constitutively transcribed.

Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter

Production of EPS I, an unusual exopolysaccharide virulence factor of the phytopathogen Pseudomonas solanacearum, requires the 18 kb eps gene cluster. DNA sequence analysis of the first seven genes of eps (epsAPBCDEF), subcellular localization of their products in maxicells, and phoA fusion analysis showed that: (i) epsA, epsB, epsE, and epsF encode exported or membrane‐associated proteins probably involved in polymerization and/or export of EPS I; (ii) epsC and epsD encode soluble enzymes probably involved in synthesis of sugar components of EPS I (N‐acetylgalactosaminuronic acid and possibly N‐acetyitrideoxygalactose, respectively); and (iii) epsP probably encodes a phosphatase involved in EPS I production in an unknown way. Non‐polar insertional mutagenesis showed that most, if not all, of these eps genes are absolutely required for production of EPS I. Using random eps::lacZ fusions and primer extension we located a transcription start site and promoter upstream of epsA. Analysis of a plasmid with this promoter fused to lacZ showed that a 140 bp regulatory region upstream of the eps transcription start site was sufficient for normal regulation of eps transcription by the multicomponent virulence gene regulatory network of P. solanacearum. Deletion of this eps promoter from a plasmid‐borne epsAPBCDE::lacZ fusion reduced its expression 10‐fold, indicating that this promoter alone is responsible for regulated transcription of an eps operon composed of at least epsAPBCDE. Analysis of genomic and plasmid‐borne eps::lacZ fusions suggested that most remaining eps genes are part of this same operon or, and this is less likely, comprise a second co‐ordinately regulated eps operon.

Inactivation of multiple virulence genes reduces the ability of Pseudomonas solanacearum to cause wilt symptoms

A logical extension of our earlier work was to determine what effect inactivating multiple virulence genes would have on the ability of P. solanacearum to wilt tomato plants. A double mutant that is EPS i and EG − was of special interest, since it should resemble the spontaneous mutant strain AW1-PC. We also wished to know whether the observed virulence of the mutants would be altered if the bacteria were required to infect plants via the roots

Comparative Genomics and an Insect Model Rapidly Identify Novel Virulence Genes of Burkholderia mallei

Burkholderia pseudomallei and its host-adapted deletion clone Burkholderia mallei cause the potentially fatal human diseases melioidosis and glanders, respectively. The antibiotic resistance profile and ability to infect via aerosol of these organisms and the absence of protective vaccines have led to their classification as major biothreats and select agents. Although documented infections by these bacteria date back over 100 years, relatively little is known about their virulence and pathogenicity mechanisms. We used in silico genomic subtraction to generate their virulome, a set of 650 putative virulence-related genes shared by B. pseudomallei and B. mallei but not present in five closely related nonpathogenic Burkholderia species. Although most of these genes are clustered in putative operons, the number of targets for mutant construction and verification of reduced virulence in animal models is formidable. Therefore, Galleria mellonella (wax moth) larvae were evaluated as a surrogate host; we found that B. pseudomallei and B. mallei, but not other phylogenetically related bacteria, were highly pathogenic for this insect. More importantly, four previously characterized B. mallei mutants with reduced virulence in hamsters or mice had similarly reduced virulence in G. mellonella larvae. Site-specific inactivation of selected genes in the computationally derived virulome identified three new potential virulence genes, each of which was required for rapid and efficient killing of larvae. Thus, this approach may provide a means to quickly identify high-probability virulence genes in B. pseudomallei, B. mallei, and other pathogens.