Using animations to teach biological processes and principles.

Well-designed animations can help students to focus on the underlying principles and processes in biology rather than relying on rote memorization. We present question-driven, terminology-free, "candymation" videos for teaching the concepts behind mitosis and meiosis as an example.

Genome-wide analysis of DNA uptake across the outer membrane of naturally competent Haemophilus influenzae.

The genomes of naturally competent Pasteurellaceae and Neisseriaceae have many short uptake sequences (USS), which allow them to distinguish self-DNA from foreign DNA. To fully characterize this preference we developed genome-wide maps of DNA uptake using both a sequence-based computational model and genomic DNA that had been sequenced after uptake by and recovery from competent Haemophilus influenzae cells. When DNA fragments were shorter than the average USS spacing of ∼1,000 bp, sharp peaks of uptake were centered at USS and separated by valleys with 1000-fold lower uptake. Long DNA fragments (1.5-17 kb) gave much less variation, with 90% of positions having uptake within 2-fold of the mean. All detectable uptake biases arose from sequences that fit the USS uptake motif. Simulated competition predicted that, in its respiratory tract environment, H. influenzae will efficiently take up its own DNA even when human DNA is present in 100-fold excess.

A competence-regulated toxin-antitoxin system in Haemophilus influenzae.

Natural competence allows bacteria to respond to environmental and nutritional cues by taking up free DNA from their surroundings, thus gaining both nutrients and genetic information. In the Gram-negative bacterium Haemophilus influenzae, the genes needed for DNA uptake are induced by the CRP and Sxy transcription factors in response to lack of preferred carbon sources and nucleotide precursors. Here we show that one of these genes, HI0659, encodes the antitoxin of a competence-regulated toxin-antitoxin operon ('toxTA'), likely acquired by horizontal gene transfer from a Streptococcus species. Deletion of the putative toxin (HI0660) restores uptake to the antitoxin mutant. The full toxTA operon was present in only 17 of the 181 strains we examined; complete deletion was seen in 22 strains and deletions removing parts of the toxin gene in 142 others. In addition to the expected Sxy- and CRP-dependent-competence promoter, HI0659/660 transcript analysis using RNA-seq identified an internal antitoxin-repressed promoter whose transcription starts within toxT and will yield nonfunctional protein. We propose that the most likely effect of unopposed toxin expression is non-specific cleavage of mRNAs and arrest or death of competent cells in the culture. Although the high frequency of toxT and toxTA deletions suggests that this competence-regulated toxin-antitoxin system may be mildly deleterious, it could also facilitate downregulation of protein synthesis and recycling of nucleotides under starvation conditions. Although our analyses were focused on the effects of toxTA, the RNA-seq dataset will be a useful resource for further investigations into competence regulation.

Evolution of Bacterial Gene Transfer Agents.

Bacterial gene transfer agents (GTAs) are small virus-like particles that package DNA fragments and inject them into cells. They are encoded by gene clusters resembling defective prophages, with genes for capsid head and tail components. These gene clusters are usually assumed to be maintained by selection for the benefits of GTA-mediated recombination, but this has never been tested. We rigorously examined the potential benefits of GTA-mediated recombination, considering separately transmission of GTA-encoding genes and recombination of all chromosomal genes. In principle GTA genes could be directly maintained if GTA particles spread them to GTA- cells often enough to compensate for the loss of GTA-producing cells. However, careful bookkeeping showed that losses inevitably exceed gains for two reasons. First, cells must lyse to release particles to the environment. Second, GTA genes are not preferentially replicated before DNA is packaged. A simulation model was then used to search for conditions where recombination of chromosomal genes makes GTA+ populations fitter than GTA- populations. Although the model showed that both synergistic epistasis and some modes of regulation could generate fitness benefits large enough to overcome the cost of lysis, these benefits neither allowed GTA+ cells to invade GTA- populations, nor allowed GTA+ populations to resist invasion by GTA- cells. Importantly, the benefits depended on highly improbable assumptions about the efficiencies of GTA production and recombination. Thus, the selective benefits that maintain GTA gene clusters over many millions of years must arise from consequences other than transfer of GTA genes or recombination of chromosomal genes.

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.

Putting my money where my mouth is: the Useful Genetics project.

The personal and societal importance of genetics has increased dramatically since the 1950s, but most introductory courses still focus on teaching students how to think like geneticists, training them in Mendelian and molecular analysis. This article is a personal account of a new course with a different goal, giving students knowledge and skills that they can use in their nonacademic lives. Useful Genetics differs from typical courses in emphasizing personal genomics, natural genetic and phenotypic variation in humans, and the consequences of genetic inheritance for breeding, inbreeding, and ancestry. Although it is a Massive Open Online Course (MOOC), taught to large global populations of learners of all ages and backgrounds, it maintains the academic rigor of a college course. The course materials are freely available for reuse by other instructors, and are being used as the foundation of a face-to-face university credit course.

Complete Genome Sequence of Haemophilus influenzae Strain 375 from the Middle Ear of a Pediatric Patient with Otitis Media.

Originally isolated from a pediatric patient with otitis media, Haemophilus influenzae strain 375 (Hi375) has been extensively studied as a model system for intracellular invasion of airway epithelial cells and other pathogenesis traits. Here, we report its complete genome sequence and methylome.

Characterization of nontypable Haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection.

Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.

Extensive cotransformation of natural variation into chromosomes of naturally competent Haemophilus influenzae.

Naturally competent bacterial species actively take up environmental DNA and can incorporate it into their chromosomes by homologous recombination. This can bring genetic variation from environmental DNA to recipient chromosomes, often in multiple long "donor" segments. Here, we report the results of genome sequencing 96 colonies of a laboratory Haemophilus influenzae strain, which had been experimentally transformed by DNA from a diverged clinical isolate. Donor segments averaged 6.9 kb (spanning several genes) and were clustered into recombination tracts of ~19.5 kb. Individual colonies had replaced from 0.1 to 3.2% of their chromosomes, and ~1/3 of all donor-specific single-nucleotide variants were present in at least one recombinant. We found that nucleotide divergence did not obviously limit the locations of recombination tracts, although there were small but significant reductions in divergence at recombination breakpoints. Although indels occasionally transformed as parts of longer recombination tracts, they were common at breakpoints, suggesting that indels typically block progression of strand exchange. Some colonies had recombination tracts in which variant positions contained mixtures of both donor and recipient alleles. These tracts were clustered around the origin of replication and were interpreted as the result of heteroduplex segregation in the original transformed cell. Finally, a pilot experiment demonstrated the utility of natural transformation for genetically dissecting natural phenotypic variation. We discuss our results in the context of the potential to merge experimental and population genetic approaches, giving a more holistic understanding of bacterial gene transfer.

Natural competence and the evolution of DNA uptake specificity.

Many bacteria are naturally competent, able to actively transport environmental DNA fragments across their cell envelope and into their cytoplasm. Because incoming DNA fragments can recombine with and replace homologous segments of the chromosome, competence provides cells with a potent mechanism of horizontal gene transfer as well as access to the nutrients in extracellular DNA. This review starts with an introductory overview of competence and continues with a detailed consideration of the DNA uptake specificity of competent proteobacteria in the Pasteurellaceae and Neisseriaceae. Species in these distantly related families exhibit strong preferences for genomic DNA from close relatives, a self-specificity arising from the combined effects of biases in the uptake machinery and genomic overrepresentation of the sequences this machinery prefers. Other competent species tested lack obvious uptake bias or uptake sequences, suggesting that strong convergent evolutionary forces have acted on these two families. Recent results show that uptake sequences have multiple "dialects," with clades within each family preferring distinct sequence variants and having corresponding variants enriched in their genomes. Although the genomic consensus uptake sequences are 12 and 29 to 34 bp, uptake assays have found that only central cores of 3 to 4 bp, conserved across dialects, are crucial for uptake. The other bases, which differ between dialects, make weaker individual contributions but have important cooperative interactions. Together, these results make predictions about the mechanism of DNA uptake across the outer membrane, supporting a model for the evolutionary accumulation and stability of uptake sequences and suggesting that uptake biases may be more widespread than currently thought.

Absence of detectable arsenate in DNA from arsenate-grown GFAJ-1 cells.

A strain of Halomonas bacteria, GFAJ-1, has been claimed to be able to use arsenate as a nutrient when phosphate is limiting and to specifically incorporate arsenic into its DNA in place of phosphorus. However, we have found that arsenate does not contribute to growth of GFAJ-1 when phosphate is limiting and that DNA purified from cells grown with limiting phosphate and abundant arsenate does not exhibit the spontaneous hydrolysis expected of arsenate ester bonds. Furthermore, mass spectrometry showed that this DNA contains only trace amounts of free arsenate and no detectable covalently bound arsenate.

Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells.

Some naturally competent bacteria exhibit both a strong preference for DNA fragments containing specific 'uptake sequences' and dramatic overrepresentation of these sequences in their genomes. Uptake sequences are often assumed to directly reflect the specificity of the DNA uptake machinery, but the actual specificity has not been well characterized for any bacterium. We produced a detailed analysis of Haemophilus influenzae's uptake specificity, using Illumina sequencing of degenerate uptake sequences in fragments recovered from competent cells. This identified an uptake motif with the same consensus as the motif overrepresented in the genome, with a 9 bp core (AAGTGCGGT) and two short flanking T-rich tracts. Only four core bases (GCGG) were critical for uptake, suggesting that these make strong specific contacts with the uptake machinery. Other core bases had weaker roles when considered individually, as did the T-tracts, but interaction effects between these were also determinants of uptake. The properties of genomic uptake sequences are also constrained by mutational biases and selective forces acting on USSs with coding and termination functions. Our findings define constraints on gene transfer by natural transformation and suggest how the DNA uptake machinery overcomes the physical constraints imposed by stiff highly charged DNA molecules.

Seventeen Sxy-dependent cyclic AMP receptor protein site-regulated genes are needed for natural transformation in Haemophilus influenzae.

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. In Haemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.

Natural transformation of Gallibacterium anatis.

Gallibacterium anatis is a pathogen of poultry. Very little is known about its genetics and pathogenesis. To enable the study of gene function in G. anatis, we have established methods for transformation and targeted mutagenesis. The genus Gallibacterium belongs to the Pasteurellaceae, a group with several naturally transformable members, including Haemophilus influenzae. Bioinformatics analysis identified G. anatis homologs of the H. influenzae competence genes, and natural competence was induced in G. anatis by the procedure established for H. influenzae: transfer from rich medium to the starvation medium M-IV. This procedure gave reproducibly high transformation frequencies with G. anatis chromosomal DNA and with linearized plasmid DNA carrying G. anatis sequences. Both DNA types integrated into the G. anatis chromosome by homologous recombination. Targeted mutagenesis gave transformation frequencies of >2 × 10(-4) transformants CFU(-1). Transformation was also efficient with circular plasmid containing no G. anatis DNA; this resulted in the establishment of a self-replicating plasmid. Nine diverse G. anatis strains were found to be naturally transformable by this procedure, suggesting that natural competence is common and the M-IV transformation procedure widely applicable for this species. The G. anatis genome is only slightly enriched for the uptake signal sequences identified in other pasteurellaceaen genomes, but G. anatis did preferentially take up its own DNA over that of Escherichia coli. Transformation by electroporation was not effective for chromosomal integration but could be used to introduce self-replicating plasmids. The findings described here provide important tools for the genetic manipulation of G. anatis.

Natural DNA uptake by Escherichia coli.

Escherichia coli has homologues of the competence genes other species use for DNA uptake and processing, but natural competence and transformation have never been detected. Although we previously showed that these genes are induced by the competence regulator Sxy as in other gamma-proteobacteria, no conditions are known that naturally induce sxy expression. We have now tested whether the competence gene homologues encode a functional DNA uptake machinery and whether DNA uptake leads to recombination, by investigating the effects of plasmid-borne sxy expression on natural competence in a wide variety of E. coli strains. High- and low-level sxy expression alone did not induce transformation in any of the strains tested, despite varying the transforming DNA, its concentration, and the incubation conditions used. Direct measurements of uptake of radiolabelled DNA were below the limit of detection, however transformants were readily detected when recombination functions were provided by the lambda Red recombinase. This is the first demonstration that E. coli sxy expression can induce natural DNA uptake and that E. coli's competence genes do encode a functional uptake machinery. However, the amount of transformation cells undergo is limited both by low levels of DNA uptake and by inefficient DNA processing/recombination.

Transformation of natural genetic variation into Haemophilus influenzae genomes.

Many bacteria are able to efficiently bind and take up double-stranded DNA fragments, and the resulting natural transformation shapes bacterial genomes, transmits antibiotic resistance, and allows escape from immune surveillance. The genomes of many competent pathogens show evidence of extensive historical recombination between lineages, but the actual recombination events have not been well characterized. We used DNA from a clinical isolate of Haemophilus influenzae to transform competent cells of a laboratory strain. To identify which of the ~40,000 polymorphic differences had recombined into the genomes of four transformed clones, their genomes and their donor and recipient parents were deep sequenced to high coverage. Each clone was found to contain ~1000 donor polymorphisms in 3-6 contiguous runs (8.1±4.5 kb in length) that collectively comprised ~1-3% of each transformed chromosome. Seven donor-specific insertions and deletions were also acquired as parts of larger donor segments, but the presence of other structural variation flanking 12 of 32 recombination breakpoints suggested that these often disrupt the progress of recombination events. This is the first genome-wide analysis of chromosomes directly transformed with DNA from a divergent genotype, connecting experimental studies of transformation with the high levels of natural genetic variation found in isolates of the same species.

Comment on "A bacterium that can grow by using arsenic instead of phosphorus".

Wolfe-Simon et al. (Research Articles, 3 June 2011, p. 1163; published online 2 December 2010) reported that bacterium GFAJ-1 can grow by using arsenic instead of phosphorus. However, the presence of contaminating phosphate in the growth medium, as well as the omission of important DNA purification steps, cast doubt on the authors' conclusion that arsenic can substitute for phosphorus in the nucleic acids of this organism.

Natural competence in strains of Actinobacillus pleuropneumoniae.

We have identified a highly transformable strain of Actinobacillus pleuropneumoniae whose competence is regulated by the competence-activator Sxy as in other Pasteurellaceae. Other strains were poorly transformable or nontransformable. The genomes of two poorly transformable strains contain intact sets of competence genes. Moreover, we show that the low competence of one of these strains is not due to an inability to induce sxy expression or to a defect in Sxy function, suggesting that some other component of the competence system is defective. Although the A. pleuropneumoniae sxy gene has only 24% identity to its Haemophilus influenzae homologue, both genes fully complemented an H. influenzae sxy knockout, demonstrating that Sxy function is conserved throughout the Pasteurellaceae.