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.

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.

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.

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.

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.

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.

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.

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.

RNA secondary structure regulates the translation of sxy and competence development in Haemophilus influenzae.

The sxy (tfoX) gene product is the central regulator of DNA uptake by naturally competent gamma-proteobacteria such as Haemophilus influenzae, Vibrio cholerae and probably Escherichia coli. However, the mechanisms regulating sxy gene expression are not understood despite being key to understanding the physiological role of DNA uptake. We have isolated mutations in H. influenzae sxy that greatly elevate translation and thus cause competence to develop in otherwise non-inducing conditions (hypercompetence). In vitro nuclease analysis confirmed the existence of an extensive secondary structure at the 5' end of sxy mRNA that sequesters the ribosome-binding site and start codon in a stem-loop. All of the hypercompetence mutations reduced mRNA base pairing, and one was shown to cause a global destabilization that increased translational efficiency. Conversely, mutations engineered to add mRNA base pairs strengthened the secondary structure, resulting in reduced translational efficiency and greatly reduced competence for genetic transformation. Transfer of wild-type cells to starvation medium improved translational efficiency of sxy while independently triggering the sugar starvation regulator (CRP) to stimulate transcription at the sxy promoter. Thus, mRNA secondary structure is responsive to conditions where DNA uptake will be favorable, and transcription of sxy is simultaneously enhanced if CRP activation signals that energy supplies are limited.

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.

Sxy induces a CRP-S regulon in Escherichia coli.

Escherichia coli is not considered naturally competent, yet it has homologues of the genes that most competent bacteria use for DNA uptake and processing. In Haemophilus influenzae and Vibrio cholerae, these genes are regulated by the Sxy and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether similar regulation occurs in E. coli. Expression of sxy strongly induced 63 transcriptional units, 34 of which required CRP for transcriptional activation and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif previously characterized in H. influenzae. As previously reported, sxy expression also induced the sigma-H regulon. Flagellar operons were downregulated by sxy expression, although motility remained unaffected. The CRP-S regulon included all of E. coli's known competence gene homologues, so we investigated Sxy's effect on competence-associated phenotypes. A sxy knockout reduced both "natural" plasmid transformation and competitive fitness in long-term culture. In addition, expression of plasmid-borne sxy led to production of type IV pilin, the main subunit of the DNA uptake machinery of most bacteria. Although H. influenzae Sxy only weakly activated the E. coli Sxy regulon, induction was dramatically improved when it was coexpressed with its cognate CRP, suggesting that intimate interactions between Sxy and CRP are required for transcriptional activation at CRP-S sites.

Non-canonical CRP sites control competence regulons in Escherichia coli and many other gamma-proteobacteria.

Escherichia coli's cAMP receptor protein (CRP), the archetypal bacterial transcription factor, regulates over a hundred promoters by binding 22 bp symmetrical sites with the consensus core half-site TGTGA. However, Haemophilus influenzae has two types of CRP sites, one like E.coli's and one with the core sequence TGCGA that regulates genes required for DNA uptake (natural competence). Only the latter 'CRP-S' sites require both CRP and the coregulator Sxy for activation. To our knowledge, the TGTGA and TGCGA motifs are the first example of one transcription factor having two distinct binding-site motifs. Here we show that CRP-S promoters are widespread in the gamma-proteobacteria and demonstrate their Sxy-dependence in E.coli. Orthologs of most H.influenzae CRP-S-regulated genes are ubiquitous in the five best-studied gamma-proteobacteria families, Enterobacteriaceae, Pasteurellaceae, Pseudomonadaceae, Vibrionaceae and Xanthomonadaceae. Phylogenetic footprinting identified CRP-S sites in the promoter regions of the Enterobacteriaceae, Pasteurellaceae and Vibrionaceae orthologs, and canonical CRP sites in orthologs of genes known to be Sxy-independent in H.influenzae. Bandshift experiments confirmed that E.coli CRP-S sequences are low affinity binding sites for CRP, and mRNA analysis showed that they require CRP, cAMP (CRP's allosteric effector) and Sxy for gene induction. This work suggests not only that the gamma-proteobacteria share a common DNA uptake mechanism, but also that, in the three best studied families, their competence regulons share both CRP-S specificity and Sxy dependence.

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.

Evolution of competence and DNA uptake specificity in the Pasteurellaceae.

BACKGROUND: Many bacteria can take up DNA, but the evolutionary history and function of natural competence and transformation remain obscure. The sporadic distribution of competence suggests it is frequently lost and/or gained, but this has not been examined in an explicitly phylogenetic context. Additional insight may come from the sequence specificity of uptake by species such as Haemophilus influenzae, where a 9 bp uptake signal sequence (USS) repeat is both highly overrepresented in the genome and needed for efficient DNA uptake. We used the distribution of competence genes and DNA uptake specificity in H. influenzae's family, the Pasteurellaceae, to examine the ancestry of competence. RESULTS: A phylogeny of the Pasteurellaceae based on 12 protein coding genes from species with sequenced genomes shows two strongly supported subclades: the Hin subclade (H. influenzae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, Mannheimia succiniciproducens, and H. somnus), and the Apl subclade (A. pleuropneumoniae, M. haemolytica, and H. ducreyi). All species contained homologues of all known H. influenzae competence genes, consistent with an ancestral origin of competence. Competence gene defects were identified in three species (H. somnus, H. ducreyi and M. haemolytica); each appeared to be of recent origin. The assumption that USS arise by mutation rather than copying was first confirmed using alignments of H. influenzae proteins with distant homologues. Abundant USS-like repeats were found in all eight Pasteurellacean genomes; the repeat consensuses of species in the Hin subclade were identical to that of H. influenzae (AAGTGCGGT), whereas members of the Apl subclade shared the consensus ACAAGCGGT. All species' USSs had the strong consensus and flanking AT-rich repeats of H. influenzae USSs. DNA uptake and competition experiments demonstrated that the Apl-type repeat is a true USS distinct from the Hin-type USS: A. pleuropneumoniae preferentially takes up DNA fragments containing the Apl-type USS over both H. influenzae and unrelated DNAs, and H. influenzae prefers its own USS over the Apl type. CONCLUSION: Competence and DNA uptake specificity are ancestral properties of the Pasteurellaceae, with divergent USSs and uptake specificity distinguishing only the two major subclades. The conservation of most competence genes over the approximately 350 million year history of the family suggests that lineages that lose competence may be evolutionary dead ends.

A novel CRP-dependent regulon controls expression of competence genes in Haemophilus influenzae.

Natural competence for DNA uptake is common among bacteria but its evolutionary function is controversial. Resolving the dispute requires a detailed understanding of both how cells decide to take up DNA and how the DNA is processed during and after uptake. We have used whole-genome microarrays to follow changes in gene expression during competence development in wild-type Haemophilus influenzae cells, and to characterize dependence of competence-induced transcription on known regulatory factors. This analysis confirmed the existence of a postulated competence regulon, characterized by a promoter-associated 22 bp competence regulatory element (CRE) closely related to the cAMP receptor protein (CRP) binding consensus. This CRE regulon contains 25 genes in 13 transcription units, only about half of which have been previously associated with competence. The new CRE genes encode a periplasmic ATP-dependent DNA ligase, homologs of SSB, RadC and the Bacillus subtilis DNA uptake protein ComEA, and eight genes of unknown function. Competence-induced transcription of genes in the CRE regulon is strongly dependent on cAMP, consistent with the known role of catabolite regulation in competence. Electrophoretic mobility-shift assays confirmed that CRE sequences are a new class of CRP-binding site. The essential competence gene sxy is induced early in competence development and is required for competence-induced transcription of CRE-regulon genes but not other CRP-regulated genes, suggesting that Sxy may act as an accessory factor directing CRP to CRE sites. Natural selection has united these 25 genes under a common regulatory mechanism. Elucidating this mechanism, and the functions of the genes, will provide a valuable window into the evolutionary function of natural competence.

Persistence and loss of meiotic recombination hotspots.

The contradiction between the long-term persistence of the chromosomal hotspots that initiate meiotic recombination and the self-destructive mechanism by which they act strongly suggests that our understanding of recombination is incomplete. This "hotspot paradox" has been reinforced by the finding that biased gene conversion also removes active hotspots from human sperm. To investigate the requirements for hotspot persistence, we developed a detailed computer simulation model of their activity and its evolutionary consequences. With this model, unopposed hotspot activity could drive strong hotspots from 50% representation to extinction within 70 generations. Although the crossing over that hotspots cause can increase population fitness, this benefit was always too small to slow the loss of hotspots. Hotspots could not be maintained by plausible rates of de novo mutation, nor by crossover interference, which alters the frequency and/or spacing of crossovers. Competition among hotspots for activity-limiting factors also did not prevent their extinction, although the rate of hotspot loss was slowed. Key factors were the probability that the initiating hotspot allele is destroyed and the nonmeiotic contributions hotspots make to fitness. Experimental investigation of these deserves high priority, because until the paradox is resolved all components of the mechanism are open to doubt.

Is quorum sensing a side effect of diffusion sensing?

Many bacteria appear to communicate by releasing and sensing autoinducer molecules, which are believed to function primarily as sensors of population density. However, this quorum-sensing hypothesis rests on very weak foundations, as neither the need for group action nor the selective conditions required for its evolution have been demonstrated. Here, I argue for a more direct function of autoinducer secretion and response - the ability to determine whether secreted molecules rapidly move away from the cell. This diffusion sensing allows cells to regulate secretion of degradative enzymes and other effectors to minimize losses owing to extracellular diffusion and mixing.

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.