Joerg Graf

Genome Sequence of Aeromonas hydrophila ATCC 7966T: Jack of All Trades

The complete genome of Aeromonas hydrophila ATCC 7966(T) was sequenced. Aeromonas, a ubiquitous waterborne bacterium, has been placed by the Environmental Protection Agency on the Contaminant Candidate List because of its potential to cause human disease. The 4.7-Mb genome of this emerging pathogen shows a physiologically adroit organism with broad metabolic capabilities and considerable virulence potential. A large array of virulence genes, including some identified in clinical isolates of Aeromonas spp. or Vibrio spp., may confer upon this organism the ability to infect a wide range of hosts. However, two recognized virulence markers, a type III secretion system and a lateral flagellum, that are reported in other A. hydrophila strains are not identified in the sequenced isolate, ATCC 7966(T). Given the ubiquity and free-living lifestyle of this organism, there is relatively little evidence of fluidity in terms of mobile elements in the genome of this particular strain. Notable aspects of the metabolic repertoire of A. hydrophila include dissimilatory sulfate reduction and resistance mechanisms (such as thiopurine reductase, arsenate reductase, and phosphonate degradation enzymes) against toxic compounds encountered in polluted waters. These enzymes may have bioremediative as well as industrial potential. Thus, the A. hydrophila genome sequence provides valuable insights into its ability to flourish in both aquatic and host environments.

Analysis, optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys.

The exploration of microbial communities by sequencing 16S rRNA genes has expanded with low-cost, high-throughput sequencing instruments. Illumina-based 16S rRNA gene sequencing has recently gained popularity over 454 pyrosequencing due to its lower costs, higher accuracy and greater throughput. Although recent reports suggest that Illumina and 454 pyrosequencing provide similar beta diversity measures, it remains to be demonstrated that pre-existing 454 pyrosequencing workflows can transfer directly from 454 to Illumina MiSeq sequencing by simply changing the sequencing adapters of the primers. In this study, we modified 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencers. Microbial communities from cows, humans, leeches, mice, sewage, and termites and a mock community were analyzed by 454 and MiSeq sequencing of the V4-V5 region and MiSeq sequencing of the V4 region. Our analysis revealed that reference-based OTU clustering alone introduced biases compared to de novo clustering, preventing certain taxa from being observed in some samples. Based on this we devised and recommend an analysis pipeline that includes read merging, contaminant filtering, and reference-based clustering followed by de novo OTU clustering, which produces diversity measures consistent with de novo OTU clustering analysis. Low levels of dataset contamination with Illumina sequencing were discovered that could affect analyses that require highly sensitive approaches. While moving to Illumina-based sequencing platforms promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.

Directed Culturing of Microorganisms Using Metatranscriptomics

ABSTRACT The vast majority of bacterial species remain uncultured, and this severely limits the investigation of their physiology, metabolic capabilities, and role in the environment. High-throughput sequencing of RNA transcripts (RNA-seq) allows the investigation of the diverse physiologies from uncultured microorganisms in their natural habitat. Here, we report the use of RNA-seq for characterizing the metatranscriptome of the simple gut microbiome from the medicinal leech Hirudo verbana and for utilizing this information to design a medium for cultivating members of the microbiome. Expression data suggested that a Rikenella-like bacterium, the most abundant but uncultured symbiont, forages on sulfated- and sialated-mucin glycans that are fermented, leading to the secretion of acetate. Histological stains were consistent with the presence of sulfated and sialated mucins along the crop epithelium. The second dominant symbiont, Aeromonas veronii, grows in two different microenvironments and is predicted to utilize either acetate or carbohydrates. Based on the metatranscriptome, a medium containing mucin was designed, which enabled the cultivation of the Rikenella-like bacterium. Metatranscriptomes shed light on microbial metabolism in situ and provide critical clues for directing the culturing of uncultured microorganisms. By choosing a condition under which the desired organism is rapidly proliferating and focusing on highly expressed genes encoding hydrolytic enzymes, binding proteins, and transporters, one can identify an organism’s nutritional preferences and design a culture medium. IMPORTANCE The number of prokaryotes on the planet has been estimated to exceed 1030 cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes. The number of prokaryotes on the planet has been estimated to exceed 1030 cells, and the overwhelming majority of them have evaded cultivation, making it difficult to investigate their ecological, medical, and industrial relevance. The application of transcriptomics based on high-throughput sequencing of RNA transcripts (RNA-seq) to microorganisms in their natural environment can provide investigators with insight into their physiologies under optimal growth conditions. We utilized RNA-seq to learn more about the uncultured and cultured symbionts that comprise the relatively simple digestive-tract microbiome of the medicinal leech. The expression data revealed highly expressed hydrolytic enzymes and transporters that provided critical clues for the design of a culture medium enabling the isolation of the previously uncultured Rikenella-like symbiont. This directed culturing method will greatly aid efforts aimed at understanding uncultured microorganisms, including beneficial symbionts, pathogens, and ecologically relevant microorganisms, by facilitating genome sequencing, physiological characterization, and genetic manipulation of the previously uncultured microbes.

Bioinformatic Genome Comparisons for Taxonomic and Phylogenetic Assignments Using Aeromonas as a Test Case

ABSTRACT Prokaryotic taxonomy is the underpinning of microbiology, as it provides a framework for the proper identification and naming of organisms. The “gold standard” of bacterial species delineation is the overall genome similarity determined by DNA-DNA hybridization (DDH), a technically rigorous yet sometimes variable method that may produce inconsistent results. Improvements in next-generation sequencing have resulted in an upsurge of bacterial genome sequences and bioinformatic tools that compare genomic data, such as average nucleotide identity (ANI), correlation of tetranucleotide frequencies, and the genome-to-genome distance calculator, or in silico DDH (isDDH). Here, we evaluate ANI and isDDH in combination with phylogenetic studies using Aeromonas, a taxonomically challenging genus with many described species and several strains that were reassigned to different species as a test case. We generated improved, high-quality draft genome sequences for 33 Aeromonas strains and combined them with 23 publicly available genomes. ANI and isDDH distances were determined and compared to phylogenies from multilocus sequence analysis of housekeeping genes, ribosomal proteins, and expanded core genes. The expanded core phylogenetic analysis suggested relationships between distant Aeromonas clades that were inconsistent with studies using fewer genes. ANI values of ≥96% and isDDH values of ≥70% consistently grouped genomes originating from strains of the same species together. Our study confirmed known misidentifications, validated the recent revisions in the nomenclature, and revealed that a number of genomes deposited in GenBank are misnamed. In addition, two strains were identified that may represent novel Aeromonas species. IMPORTANCE Improvements in DNA sequencing technologies have resulted in the ability to generate large numbers of high-quality draft genomes and led to a dramatic increase in the number of publically available genomes. This has allowed researchers to characterize microorganisms using genome data. Advantages of genome sequence-based classification include data and computing programs that can be readily shared, facilitating the standardization of taxonomic methodology and resolving conflicting identifications by providing greater uniformity in an overall analysis. Using Aeromonas as a test case, we compared and validated different approaches. Based on our analyses, we recommend cutoff values for distance measures for identifying species. Accurate species classification is critical not only to obviate the perpetuation of errors in public databases but also to ensure the validity of inferences made on the relationships among species within a genus and proper identification in clinical and veterinary diagnostic laboratories. Improvements in DNA sequencing technologies have resulted in the ability to generate large numbers of high-quality draft genomes and led to a dramatic increase in the number of publically available genomes. This has allowed researchers to characterize microorganisms using genome data. Advantages of genome sequence-based classification include data and computing programs that can be readily shared, facilitating the standardization of taxonomic methodology and resolving conflicting identifications by providing greater uniformity in an overall analysis. Using Aeromonas as a test case, we compared and validated different approaches. Based on our analyses, we recommend cutoff values for distance measures for identifying species. Accurate species classification is critical not only to obviate the perpetuation of errors in public databases but also to ensure the validity of inferences made on the relationships among species within a genus and proper identification in clinical and veterinary diagnostic laboratories.

Evolutionary and Diagnostic Implications of Intragenomic Heterogeneity in the 16S rRNA Gene in Aeromonas Strains

Sequencing 16S rRNA genes (SSU) cloned from Aeromonas strains revealed that strains contained up to six copies differing by < or = 1.5%. The SSU copies from Aeromonas veronii LMG13695 clustered with sequences from four Aeromonas species. These results demonstrate intragenomic heterogeneity of SSU and suggest caution when using SSU to identify aeromonads.

Culture-Independent Characterization of the Digestive-Tract Microbiota of the Medicinal Leech Reveals a Tripartite Symbiosis

ABSTRACT Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the α-, γ-, and δ-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.

Interaction between innate immune cells and a bacterial type III secretion system in mutualistic and pathogenic associations.

Animals house a community of bacterial symbionts in their digestive tracts that contribute to their well being. The medicinal leech, Hirudo verbana, has a remarkably simple gut population carrying two extracellular microbes in the crop where the ingested blood is stored. This simplicity renders it attractive for studying colonization factors. Aeromonas veronii, one of the leech symbionts, can be genetically manipulated and is a pathogen of mammals. Screening transposon mutants of A. veronii for colonization defects in the leech, we found one mutant, JG752, with a transposon insertion in an ascU homolog, encoding an essential component of type III secretion systems (T3SS). Competing JG752 against the wild type revealed that JG752 was increasingly attenuated over time (10-fold at 18 h and >10,000-fold at 96 h). This colonization defect was linked to ascU by complementing JG752 with the operon containing ascU. Fluorescence in situ hybridization analysis revealed that at 42 h 38% of JG752 cells were phagocytosed by leech macrophage-like cells compared with <0.1% of the parental strain. Using mammalian macrophages, a lactate dehydrogenase release assay revealed that cytotoxicity was significantly reduced in macrophages exposed to JG752. In a mouse septicemia model, JG752 killed only 30% of mice, whereas the parent strain killed 100%, showing the importance of T3SS for both pathogenesis and mutualism. Phagocytic immune cells are important not only in defending against pathogens but also in maintaining the mutualistic symbiont community inside the leech, demonstrating that animals use similar, conserved mechanisms to control bacterial populations, even when the outcomes differ dramatically.

Novel effects of a transposon insertion in the Vibrio fischeri glnD gene: defects in iron uptake and symbiotic persistence in addition to nitrogen utilization

Vibrio fischeri is the sole species colonizing the light‐emitting organ of the Hawaiian squid, Euprymna scolopes. Upon entering the nascent light organ of a newly hatched juvenile squid, the bacteria undergo morphological and physiological changes that include the loss of flagellation and the induction of bioluminescence. These and other events reveal a pattern of genetic regulation that is a response to the colonization of host tissue. In this study, we isolated and characterized a glnD::mTn5Cm mutant of V. fischeri. In addition to the predicted defects in the efficiency of nitrogen utilization, this glnD mutant had an unexpected reduction in the ability to produce siderophore and grow under iron‐limiting conditions. Although the glnD mutant could colonize juvenile squid normally over the first 24 h, it was subsequently unable to persist in the light organ to the usual extent. This persistence phenotype was more severe if the mutant was pregrown under iron‐limiting conditions before inoculation, but could be ameliorated by the presence of excess iron. These results indicate that the ability to respond to iron limitation may be an important requirement in the developing symbiosis. Supplying the glnD gene in trans restored normal efficiency of nitrogen use, iron sequestration and colonization phenotypes to the glnD::mTn5Cm mutant; thus, there appears to be a genetic and/or metabolic linkage between nitrogen sensing, siderophore synthesis and symbiosis competence in V. fischeri that involves the glnD gene.

Spatial and temporal population dynamics of a naturally occurring two-species microbial community inside the digestive tract of the medicinal leech.

ABSTRACT The medicinal leech, Hirudo verbana, is one of the simplest naturally occurring models for digestive-tract symbioses, where only two bacterial species, Aeromonas veronii bv. sobria (γ-Proteobacteria) and a Rikenella-like bacterium (Bacteroidetes), colonize the crop, the largest compartment of the leech digestive tract. In this study, we investigated spatial and temporal changes of the localization and microcolony structure of the native symbionts in the crop, after ingestion of a sterile blood meal, by fluorescence in situ hybridization. The population dynamics differed between the two symbiotic bacteria. A. veronii was detected mainly as individual cells inside the intraluminal fluid (ILF) during 14 days after feeding (daf) unless it was found in association with Rikenella microcolonies. The Rikenella-like bacteria were observed not only inside the ILF but also in association with the luminal surface of the crop epithelium. The sizes of Rikenella microcolonies changed dynamically through the 14-day period. From 3 daf onward, mixed microcolonies containing both species were frequently observed, with cells of both species tightly associating with each other. The sizes of the mixed microcolonies were consistently larger than the size of either single-species microcolony, suggesting a synergistic interaction of the symbionts. Lectin staining with succinylated wheat germ agglutinin revealed that the planktonic microcolonies present in the ILF were embedded in a polysaccharide matrix containing N-acetylglucosamine. The simplicity, symbiont-symbiont interaction, and mixed microcolonies of this naturally occurring, digestive-tract symbiosis lay the foundation for understanding the more complex communities residing in most animals.

Complex Evolutionary History of the Aeromonas veronii Group Revealed by Host Interaction and DNA Sequence Data

Aeromonas veronii biovar sobria, Aeromonas veronii biovar veronii, and Aeromonas allosaccharophila are a closely related group of organisms, the Aeromonas veronii Group, that inhabit a wide range of host animals as a symbiont or pathogen. In this study, the ability of various strains to colonize the medicinal leech as a model for beneficial symbiosis and to kill wax worm larvae as a model for virulence was determined. Isolates cultured from the leech out-competed other strains in the leech model, while most strains were virulent in the wax worms. Three housekeeping genes, recA, dnaJ and gyrB, the gene encoding chitinase, chiA, and four loci associated with the type three secretion system, ascV, ascFG, aexT, and aexU were sequenced. The phylogenetic reconstruction failed to produce one consensus tree that was compatible with most of the individual genes. The Approximately Unbiased test and the Genetic Algorithm for Recombination Detection both provided further support for differing evolutionary histories among this group of genes. Two contrasting tests detected recombination within aexU, ascFG, ascV, dnaJ, and gyrB but not in aexT or chiA. Quartet decomposition analysis indicated a complex recent evolutionary history for these strains with a high frequency of horizontal gene transfer between several but not among all strains. In this study we demonstrate that at least for some strains, horizontal gene transfer occurs at a sufficient frequency to blur the signal from vertically inherited genes, despite strains being adapted to distinct niches. Simply increasing the number of genes included in the analysis is unlikely to overcome this challenge in organisms that occupy multiple niches and can exchange DNA between strains specialized to different niches. Instead, the detection of genes critical in the adaptation to specific niches may help to reveal the physiological specialization of these strains.