Stephanus N. Venter

Pantoea ananatis: an unconventional plant pathogen

UNLABELLED Pantoea ananatis causes disease symptoms in a wide range of economically important agricultural crops and forest tree species worldwide. It is regarded as an emerging pathogen based on the increasing number of reports of diseases occurring on previously unrecorded hosts in different parts of the world. Its unconventional nature lies in the fact that, unlike the majority of plant pathogenic microbes, P. ananatis is capable of infecting humans and occurs in diverse ecological niches, such as part of a bacterial community contaminating aviation jet fuel tanks and contributing to growth promotion in potato and pepper. TAXONOMY Bacteria; Gammaproteobacteria; family Enterobacteriaceae; genus Pantoea. MICROBIOLOGICAL PROPERTIES Gram-negative; facultatively anaerobic; most strains are motile and produce a yellow pigment in culture; indole positive. BIOLOGY: Pantoea ananatis is a common epiphyte; it also occurs endophytically in hosts where it has been reported to cause disease symptoms and in hosts where no such symptoms have been described. Some strains are ice-nucleating, a feature which has been used as a biological control mechanism against some insect pests of agricultural crops and by the food industry. DISEASE SYMPTOMS Pantoea ananatis infects both monocotyledonous and dicotyledonous plants. The symptoms are diverse depending on the host infected, and include leaf blotches and spots, die-back, and stalk, fruit and bulb rot. BIOLOGICAL CONTROL AGENT: Pantoea ananatis has both antifungal and antibacterial properties. These characteristics have the potential of being exploited by biological control specialists.

A sensitive seminested PCR method for the detection of Shigella in spiked environmental water samples

A rapid seminested polymerase chain reaction (PCR) method for the specific, sensitive detection of virulent Shigella spp. in spiked environmental water samples was developed. A set of primers specific for the invasion plasmid antigen gene (ipaH) of virulent Shigella spp. and enteroinvasive Escherichia coli produced a 620-bp fragment that was used as template for the seminested primer pair delineating a 401-bp fragment. By using agarose gel electrophoresis for detection of the seminested PCR-amplified products, a detection limit of 1.6 x 10(3) cfu S. flexneri was obtained with amplification reactions from crude bacterial lysates. The PCR procedure coupled with an enrichment culture incubated for 6 h detected as few as 1.6 S. flexneri organisms in pure culture. Treated sewage, ground, surface and drinking water samples collected from various sources were seeded with S. flexneri and incubated in GN broth for 6 h before detection by seminested PCR. A detection limit lower than 14 cfu/ml was achieved in some water samples. The results indicate that the described seminested PCR has the advantage of a rapid turnaround time and it fulfills the requirements of sensitivity and specificity for use in an environmental laboratory.

Pantoea vagans sp. nov., Pantoea eucalypti sp. nov., Pantoea deleyi sp. nov. and Pantoea anthophila sp. nov.

Bacteria isolated from eucalyptus leaves and shoots showing symptoms of blight and die-back collected in Uganda, Uruguay and Argentina and from maize displaying brown stalk rot symptoms in South Africa were tentatively placed in the genus Pantoea on the basis of phenotypic and biochemical tests. These isolates, together with two strains (LMG 2558 and LMG 2560) previously assigned to Pantoea agglomerans based on protein electrophoregrams but later excluded from this species, were further investigated using molecular techniques. 16S rRNA gene sequencing and multilocus sequence analyses (MLSA) revealed that the strains were phylogenetically closely related to Pantoea agglomerans, Pantoea stewartii and Pantoea ananatis. MLSA and amplified fragment length polymorphism analysis placed the strains into four separate clusters, not containing any of the type strains of species of the genus Pantoea. DNA-DNA hybridization confirmed the classification of the isolates into four novel species, for which the names Pantoea vagans sp. nov. (type strain R-21566T=LMG 24199T=BCC 105T=BD 765T), Pantoea eucalypti sp. nov. (type strain R-25678T=LMG 24197T=BCC 076T=BD 769T), Pantoea deleyi sp. nov. (type strain R-31523T=LMG 24200T=BCC 109T=BD 767T) and Pantoea anthophila sp. nov. (type strain LMG 2558T=BD 871T=NCPPB 1682T) are proposed.

Genome Sequence of Pantoea ananatis LMG20103, the Causative Agent of Eucalyptus Blight and Dieback

Pantoea ananatis is a Gram-negative plant pathogen that causes disease on a broad range of host plants, including pineapple, maize, rice, onion, melons, and Eucalyptus, and has been implicated in several cases of human disease. Here, we report the genome sequence of P. ananatis LMG20103 isolated from diseased Eucalyptus in South Africa.

Comparative genomics of the type VI secretion systems of Pantoea and Erwinia species reveals the presence of putative effector islands that may be translocated by the VgrG and Hcp proteins

BackgroundThe Type VI secretion apparatus is assembled by a conserved set of proteins encoded within a distinct locus. The putative effector proteins Hcp and VgrG are also encoded within these loci. We have identified numerous distinct Type VI secretion system (T6SS) loci in the genomes of several ecologically diverse Pantoea and Erwinia species and detected the presence of putative effector islands associated with the hcp and vgrG genes.ResultsBetween two and four T6SS loci occur among the Pantoea and Erwinia species. While two of the loci (T6SS-1 and T6SS-2) are well conserved among the various strains, the third (T6SS-3) locus is not universally distributed. Additional orthologous loci are present in Pantoea sp. aB-valens and Erwinia billingiae Eb661. Comparative analysis of the T6SS-1 and T6SS-3 loci showed non-conserved islands associated with the vgrG and hcp, and vgrG genes, respectively. These regions had a G+C content far lower than the conserved portions of the loci. Many of the proteins encoded within the hcp and vgrG islands carry conserved domains, which suggests they may serve as effector proteins for the T6SS. A number of the proteins also show homology to the C-terminal extensions of evolved VgrG proteins.ConclusionsExtensive diversity was observed in the number and content of the T6SS loci among the Pantoea and Erwinia species. Genomic islands could be observed within some of T6SS loci, which are associated with the hcp and vgrG proteins and carry putative effector domain proteins. We propose new hypotheses concerning a role for these islands in the acquisition of T6SS effectors and the development of novel evolved VgrG and Hcp proteins.

Identification methods for Legionella from environmental samples

Laboratories responsible for Legionella diagnostics around the world use a number of different culturing methods of non-equivalent sensitivities and specificities, to detect Legionella species in environmental samples. Specific countries usually standardize and use one approved method. For example, laboratories in Australia use the Australian Standard (AS) method and those in Europe, the International Standard method (ISO). However, no standard culturing methods have been established in South Africa to date. As a result, there is uncertainty about the true prevalence and most common species of Legionella present in the South African environment. In an attempt to provide guidelines for the development of a standard method specific for South Africa, the ISO, AS and a most probable number method were evaluated and compared. In addition, the effect of sample re-incubation with autochthonous amoebae on culture outcome was studied. Samples were collected from four environments, representing industrial water, mine water and biofilm. The samples were concentrated by membrane filtration and divided into three portions and cultured without pretreatment, after acid treatment and after heat treatment, on four culture media namely alphaBCYE, BMPA, MWY and GVPC agar. A selective approach, incorporating heat treatment, but not acid treatment, combined with culture on alphaBCYE and GVPC or MWY, was most appropriate for legionellae detection in the samples evaluated. Legionellae were cultured from 82% of the environmental samples we evaluated. In 54% of the samples tested, legionellae were present in numbers equal to or exceeding 10(2) colony-forming units per milliliter (cfu/ml). Legionella pneumophila serogroups (SGs) 1-14 were the most prevalent species and were present as single, or a combination of two or more SGs in a number of samples tested. Re-incubation of sample concentrates with autochthonous amoebae improved the culturability of legionellae in 50% of cultures on alphaBCYE and 25% on GVPC.

Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation-pit-stop semi-nested PCR procedure.

A pit‐stop semi‐nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml−1 was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit‐stop semi‐nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples.

Analysis of the Pantoea ananatis pan-genome reveals factors underlying its ability to colonize and interact with plant, insect and vertebrate hosts

BackgroundPantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms.ResultsThe pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors.ConclusionsP. ananatis has an ‘open’ pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.

South African papilionoid legumes are nodulated by diverse Burkholderia with unique nodulation and nitrogen-fixation Loci

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.

Metagenomic analysis of bacterial diversity of Siloam hot water spring, Limpopo, South Africa

The bacterial diversity of Siloam hot water spring was determined using 454 pyrosequencing of two 16S rRNA variable regions V1-3 and V4-7. Analysis of the community DNA revealed that the phyla Proteobacteria, Cyanobacteria, Bacteriodetes, Planctomycetes, Firmicutes, Chloroflexi and Verrucomicrobia were the most abundant. The bacterial diversity detectable and classifiable was greater when the V4-7 variable region was used compared to the V1-3 region. The most abundant bacteria genera detected with region V1-3 were; Stenotrophomonas (23.3%), Aquaspirillum (5.11%) , Zavarzinella (2.73%), Haliscomenobacteria (1.25%), Rheinheimera (1.14%) and Tepidomonas (1.14%). All the other detectable genera were below 0.6%. Genera detected with region V4-7 from most abundant were; Stenotrophomonas (17.96%), Zavarzinella (5.81%), Aquaspirillum (4.75%), Rheinheimera (3.52%), GPI (1.41%), Gemmata (1.41%) and Syntrophobacter (1.06%). All the other genera detected were below 0.7%. Siloam is one of the hottest thermal springs in South Africa (63°C), the water has a pH of 9.5 and is relatively high in fluoride and bromide; it is possible that the physicochemical properties could have some influence on the diversity of bacteria. This article reports on the first phylogenetic analysis of a South African thermal spring bacterial community. Key words : Thermophilic, hot springs, biodiversity, 454 sequencing, South Africa.