Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences

Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.     

Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.     

The Response regulator HrpY of Dickeya dadantii 3937 regulates virulence genes not linked to the hrp cluster

HrpX/Y is a putative two-component system (TCS) encoded within the type III secretion system (T3SS) gene cluster of Dickeya dadantii. A linear regulatory cascade initiated by HrpX/Y that leads to activation of the downstream T3SS genes via HrpS and HrpL was described previously. Therefore, in D. dadantii, HrpX/Y plays an important role in regulation of genes involved in bacteria-plant interactions and bacterial aggregation via the T3SS. HrpX/Y is the only TCS shared among the plant-pathogenic enterobacteria that is not also present in animal-associated enterobacteria. To date, the genes known to be regulated by HrpY are restricted to the hrp and hrc genes and no signal has been identified that triggers HrpY-dependent gene expression. We demonstrated that HrpY interacts with the hrpS promoter in vitro. We then used a transposon-based system to isolate previously unidentified HrpY-dependent genes, including genes previously shown to affect virulence, including kdgM and acsC. HrpY is a dual regulator, positively regulating at least 10 genes in addition to those in the hrp gene cluster and negatively regulating at least 5 genes. The regulatory effect on one gene depended on the culture medium used. Of the 16 HrpY-regulated genes identified in this screen, 14 are not present in Pectobacterium atrosepticum, the nearest relative of D. dadantii with a sequenced genome. None of the newly identified HrpY-regulated genes were required for bacterial aggregation; thus, neither acyl-homoserine lactone-mediated quorum sensing nor the Rcs signal transduction system which regulates colanic acid, a molecule that plays an important role in biofilm formation in other enterobacteria, are required for D. dadantii aggregation.     

Classification of plant associated bacteria using RIF, a computationally derived DNA marker

A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.     

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

The 3-hydroxy-2-butanone pathway is required for Pectobacterium carotovorum pathogenesis

Pectobacterium species are necrotrophic bacterial pathogens that cause soft rot diseases in potatoes and several other crops worldwide. Gene expression data identified Pectobacterium carotovorum subsp. carotovorum budB, which encodes the α-acetolactate synthase enzyme in the 2,3-butanediol pathway, as more highly expressed in potato tubers than potato stems. This pathway is of interest because volatiles produced by the 2,3-butanediol pathway have been shown to act as plant growth promoting molecules, insect attractants, and, in other bacterial species, affect virulence and fitness. Disruption of the 2,3-butanediol pathway reduced virulence of P. c. subsp. carotovorum WPP14 on potato tubers and impaired alkalinization of growth medium and potato tubers under anaerobic conditions. Alkalinization of the milieu via this pathway may aid in plant cell maceration since Pectobacterium pectate lyases are most active at alkaline pH.     

Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster

The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue. Some soft-rot Erwinia spp. also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system. The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp. is not clear. We isolated and characterized 11 hrp genes of Erwinia carotovora subsp. carotovora. Three putative sigmaL-dependent Hrp box promoter sequences were found. The genes were expressed when the bacteria were grown in Hrp-inducing medium. The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes. An E. carotovora strain with mutated hrcC, an essential hrp gene, was constructed. The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection.     

Niche-specificity and the variable fraction of the Pectobacterium pan-genome

We compare genome sequences of three closely related soft-rot pathogens that vary in host range and geographical distribution to identify genetic differences that could account for lifestyle differences. The isolates compared, Pectobacterium atrosepticum SCRI1043, P. carotovorum WPP14, and P. brasiliensis 1692, represent diverse lineages of the genus. P. carotovorum and P. brasiliensis genome contigs, generated by 454 pyrosequencing ordered by reference to the previously published complete circular chromosome of P. atrosepticum genome and each other, account for 96% of the predicted genome size. Orthologous proteins encoded by P. carotovorum and P. brasiliensis are approximately 95% identical to each other and 92% identical to P. atrosepticum. Multiple alignment using Mauve identified a core genome of 3.9 Mb conserved among these Pectobacterium spp. Each core genome is interrupted at many points by species-specific insertions or deletions (indels) that account for approximately 0.9 to 1.1 Mb. We demonstrate that the presence of a hrpK-like type III secretion system-dependent effector protein in P. carotovorum and P. brasiliensis and its absence from P. atrosepticum is insufficient to explain variability in their response to infection in a plant. Additional genes that vary among these species include those encoding peptide toxin production, enzyme production, secretion proteins, and antibiotic production, as well as differences in more general aspects of gene regulation and metabolism that may be relevant to pathogenicity.     

Identification by Tn-seq of Dickeya dadantii genes required for survival in chicory plants

The identification of the virulence factors of plant-pathogenic bacteria has relied on the testing of individual mutants on plants, a time-consuming process. Transposon sequencing (Tn-seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft-rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant–bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆carA, ∆purF, ∆purL, ∆guaB and ∆pyrE were unable to survive in the plant in contrast with the wild-type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆rsmC and ∆gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.     

SlyA regulates type III secretion system (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937

The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.     

The Rcs phosphorelay modulates the expression of plant cell wall degrading enzymes and virulence in Pectobacterium carotovorum ssp. carotovorum

Production of plant cell wall degrading enzymes, the major virulence factors of soft-rot Pectobacterium species, is controlled by many regulatory factors. Pectobacterium carotovorum ssp. carotovorum SCC3193 encodes an Rcs phosphorelay system that involves two sensor kinases, RcsC(Pcc) and RcsD(Pcc), and a response regulator RcsB(Pcc) as key components of this system, and an additional small lipoprotein RcsF(Pcc). This study indicates that inactivation of rcsC(Pcc), rcsD(Pcc) and rcsB(Pcc) enhances production of virulence factors with the highest effect detected for rcsB(Pcc). Interestingly, mutation of rcsF(Pcc) has no effect on virulence factors synthesis. These results suggest that in SCC3193 a parallel phosphorylation mechanism may activate the RcsB(Pcc) response regulator, which acts as a repressor suppressing the plant cell wall degrading enzyme production. Enhanced production of virulence factors in Rcs mutants is more pronounced when bacteria are growing in the absence of plant signal components.     

Butanediol production by Aerobacter aerogenes NRRL B199: effects of initial substrate concentration and aeration agitation

Butanediol production by Aerobacter aerogenes NRRL B199 grown on glucose requires an optimal rate of aeration for the obtention of butanediol 2, 3. In the absence of air, Aerobacter aerogenes NRRL B199 growth and production are weak. Agitation-aeration is necessary for producing the biomass, but an excess of oxygen proves to be toxic with regard to metabolite production. Oxygen is a limiting substrate with regard to growth and an inhibitor with regard to the specific metabolite productivity. This observation is discussed from a kinetic stand point and in relation to the search for the optimum oxygen transfer coefficient (K(L)a), which is found to be in the range of 50-100h(-1). It has also been observed that K(L)a increases during the fermentation cycle. The initial substrate concentration effects the yield production of biomass and butanediol production. Low yields of butanediol are obtained at low initial sugar concentrations, but good yields of butanediol are obtained (0.45 g/g) at high concentrations of glucose (195 g/L). Carbon substrates and butanediol are inhibitors of cell growth while butanediol is not quite an inhibitor of the specific rate of butanediol production for the range of butanediol of 0-100 g/L.