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A new workshop brings together under-represented participants to gain experience working in diverse teams, identifying problems worth solving, and learning evidence-based entrepreneurship approaches.

The physical impacts of climate change have been widely addressed in scientific journals and the media. These include extreme weather events, sea level rise, ocean acidification, melting of ice sheets and glaciers, and many other responses to the continuing increase in the average global temperature. The media has also emphasized the impact of these events on climate migration, heat stress, and allergies. However, the conversation rarely addresses the impact of climate change on microbes and the ensuing impact on human health. Microbes can both accelerate and mitigate the drivers of climate change. Climate change can also exacerbate infectious diseases caused by microbes. This chapter provides some examples of how the varied physical impacts of climate change modulate the roles of microbes play in the health of humans, animals, and plants.

Oscillations are ubiquitous features of brain dynamics that undergo task-related changes in synchrony, power, and frequency. The impact of those changes on target networks is poorly understood. In this work, we used a biophysically detailed model of prefrontal cortex (PFC) to explore the effects of varying the spike rate, synchrony, and waveform of strong oscillatory inputs on the behavior of cortical networks driven by them. Interacting populations of excitatory and inhibitory neurons with strong feedback inhibition are inhibition-based network oscillators that exhibit resonance (i.e., larger responses to preferred input frequencies). We quantified network responses in terms of mean firing rates and the population frequency of network oscillation; and characterized their behavior in terms of the natural response to asynchronous input and the resonant response to oscillatory inputs. We show that strong feedback inhibition causes the PFC to generate internal (natural) oscillations in the beta/gamma frequency range (>15 Hz) and to maximize principal cell spiking in response to external oscillations at slightly higher frequencies. Importantly, we found that the fastest oscillation frequency that can be relayed by the network maximizes local inhibition and is equal to a frequency even higher than that which maximizes the firing rate of excitatory cells; we call this phenomenon population frequency resonance. This form of resonance is shown to determine the optimal driving frequency for suppressing responses to asynchronous activity. Lastly, we demonstrate that the natural and resonant frequencies can be tuned by changes in neuronal excitability, the duration of feedback inhibition, and dynamic properties of the input. Our results predict that PFC networks are tuned for generating and selectively responding to beta- and gamma-rhythmic signals due to the natural and resonant properties of inhibition-based oscillators. They also suggest strategies for optimizing transcranial stimulation and using oscillatory networks in neuromorphic engineering. Author Summary The prefrontal cortex (PFC) flexibly encodes task-relevant representations and outputs biases to mediate higher cognitive functions. The relevant neural ensembles undergo task-related changes in oscillatory dynamics at beta- and gamma frequencies. Using a computational model of the PFC network, we show that strong feedback inhibition causes the PFC to generate internal oscillations and to prefer external oscillations at similar frequencies. The precise frequencies that are generated and preferred can be flexibly tuned by varying the synchrony and strength of input network activity, the level of background excitation, and neuromodulation of intrinsic ion currents. We also show that the peak output frequency in response to external oscillations, which depends on the synchrony and strength of the input as well as the strong inhibitory feedback, is faster than the internally generated frequency, and that this difference enables exclusive response to oscillatory inputs. These properties enable changes in oscillatory dynamics to govern the selective processing and gating of task-relevant signals in service of cognitive control.

Bacteria and their viruses (bacteriophages or phages) have a complex relationship that is in constant flux. Studies in natural environments, and by manipulations of phages and bacteria in a laboratory setting, have demonstrated that phages can profoundly influence bacterial populations. Phages can alter the density of different bacteria by lytic infection, and the genetic makeup of bacteria can be altered by lysogeny or transduction. Phages are promiscuous mediators of genetic exchange and often carry genes capable of altering the phenotypes of their bacterial hosts. The genetic traits acquired from phages can influence adaptation of bacteria to an environment by providing enhanced or novel metabolic properties, resistance to other phages or protozoan predators, and acquisition of antibiotic resistance or new virulence traits. The improvement in sequencing technology that sparked the microbial metagenomic revolution has provided another tool for understanding the impact of phages on bacterial populations. Metagenomic analysis of virus populations (“viromics”) has provided insight into the surprising extent that phages modulate the bacterial genome. While the benefit to the phages for transferring novel properties to its host is often poorly understood, this relationship clearly provides a selective advantage because these properties are maintained in many environments and including otherwise adverse conditions for the bacterial host. In this chapter, we will discuss how phages influence the fitness of bacteria in particular environmental niches.

Recent advances in metagenomics research have generated a bounty of information that provides insight into the dynamic genetic exchange occurring between bacteriophage (phage) and their bacterial hosts. Metagenomic studies of the microbiomes from a variety of environments have shown that many of the genes sequenced are of phage origin. Among these genes are phage-encoded exotoxin genes. When phage that carry these genes infect an appropriate bacterial host, the bacterium undergoes lysogenic conversion, converting the bacterium from an avirulent strain to a pathogen that can cause human disease. Transfer of the exotoxin genes between bacteria has been shown to occur in marine environments, animal and human intestines and sewage treatment plants. Surprisingly, phage that encode exotoxin genes are commonly found in environments that lack the cognate bacteria commonly associated with the specific toxin-mediated disease and have been found to be associated with alternative environmental bacterial hosts. These findings suggest that the exotoxin genes may play a beneficial role for the bacterial host in nature, and that this environmental reservoir of exotoxin genes may play a role in the evolution of new bacterial pathogens.

Microbes account for more than 90% of the cells of the human maintenance of organ alloengraftment has body, forming distinctive communities at different sites. These communities affect health—promoting organ development, stimulating the immune system, providing nutrients, and excluding pathogens. These communities cause pathologies, including periodontal disease, ulcers, and secondary pneumonias, while influencing other conditions, including obesity and atherosclerosis.

One Health is a paradigm that encompasses the health of humans, animals, and their environment, recognizing that the health of each domain is inextricably interconnected. The One Health concept is interdisciplinary, comprising, among others, public health, clinical medicine, microbiology, ecology, and geography, as well as public outreach. One Health has particularly great relevance for microbiology as a unifying discipline that connects health among humans, animals, and the environment. Given that over 60% of emerging infectious disease events are caused by the transmission of an infectious agent from animals (zoonoses), with 75% of these originating from wildlife, employing a systematic One Health approach has great potential for reducing threats to global health from infectious diseases. The One Health approach can advance health care for the 21st century and beyond by accelerating biomedical research, enhancing public health efficacy, expeditiously expanding the scientific knowledge base, and improving medical education and clinical care.

There are significant opportunities for improving human, animal, and environmental health by adopting a One Health approach. One Health approaches are likely to have a major impact on public health, with a focus on surveillance and upstream interventions that are likely to reap obvious and rapid benefits for the health of human populations. However, despite the obvious benefits, the barriers to achieving a comprehensive One Health approach are formidable given that education, research, diagnostics, surveillance, and funding for human medicine, veterinary medicine, and environmental health often exist as separate silos with limited exchange. These barriers must be overcome if the benefits of One Health are to be realized.

Global climate change can alter the distribution of microbial pathogens and vectors that transmit infectious diseases, exposing humans to newly emerging or reemerging diseases. Early detection of potential pathogens and vectors in the environment can facilitate upstream interventions that limit the spread of infectious disease. Metagenomics is the analysis of DNA sequences from a population of microorganisms in a particular environment, followed by the computational reconstruction of the data to determine what organisms are present and predict their role in the environment. Defining the microbial populations associated with humans, animals, and their environment provides insight into the structure of microbial communities in any particular niche, including the abundance, diversity, and composition of the microbes and viruses present. It can also reveal the distribution of virulence genes within that niche. These data can be used to identify reservoirs of pathogens in an environment and predict environments with a high probability for evolution of new pathogens or outbreaks caused by known pathogens, thereby facilitating approaches to prevent infections of animals or humans before serious outbreaks of infectious disease.

There are >2,600 recognized serovars of Salmonella enterica . Many of these Salmonella serovars have a broad host range and can infect a wide variety of animals, including mammals, birds, reptiles, amphibians, fish, and insects. In addition, Salmonella can grow in plants and can survive in protozoa, soil, and water. Hence, broad-host-range Salmonella can be transmitted via feces from wild animals, farm animals, and pets or by consumption of a wide variety of common foods: poultry, beef, pork, eggs, milk, fruit, vegetables, spices, and nuts. Broad-host-range Salmonella pathogens typically cause gastroenteritis in humans. Some Salmonella serovars have a more restricted host range that is associated with changes in the virulence plasmid pSV, accumulation of pseudogenes, and chromosome rearrangements. These changes in host-restricted Salmonella alter pathogen-host interactions such that host-restricted Salmonella organisms commonly cause systemic infections and are transmitted between host populations by asymptomatic carriers. The secondary consequences of efforts to eliminate host-restricted Salmonella serovars demonstrate that basic ecological principles govern the environmental niches occupied by these pathogens, making it impossible to thwart Salmonella infections without a clear understanding of the human, animal, and environmental reservoirs of these pathogens. Thus, transmission of S. enterica provides a compelling example of the One Health paradigm because reducing human infections will require the reduction of Salmonella in animals and limitation of transmission from the environment.

Most Salmonella serovars are general pathogens that infect a variety of hosts. These “generalist” serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.

As the number of completed genome sequences increases, there is increasing emphasis on comparative genomic analysis of closely related organisms. Comparison of the similarities and differences between the five publicly available Salmonella genome sequences reveals extensive sequence conservation among the Salmonella serovars. However, horizontal gene transfer has provided each genome with between 10% and 12% of unique DNA. Genome comparisons of the closely related salmonellae emphasize the insights that can be gleaned from sequencing genomes of a single species.

Bacterial genetics has been strongly influenced by the work of John Roth and his laboratory. Work in John’s laboratory has covered a large number of topics, beginning with the genetic regulation of the histidine (his) biosynthetic operon and extending to many other metabolic pathways and genetic processes. John became interested in bacterial genetics while an undergraduate at Harvard University. The regulatory mutations were scattered widely around the genome and affected functions involved in translation (e.g., histidyl-tRNA synthetase, tRNAHis, and tRNA modifying and processing enzymes). Interest in genetics of tRNA led John’s lab to work on a variety of informational suppressors, including recessive nonsense suppressors and many classes of frameshift suppressors in which altered tRNAs caused translation to shift reading phase. John became interested in chromosomal duplications during early studies on nonsense suppressor tRNAs. Suppressor mutations that alter an essential tRNA type are lethal unless they arise in one copy of a preexisting duplication of the tRNA gene. The genetic tools developed in John’s lab helped change the perspectives on the pulsating rhythms of chromosome organization. Other work led to ideas on the evolution of bacterial operons by horizontal transfer and origins of new genes by selective amplification. In addition to his research accomplishments, John is an enthusiastic, stimulating speaker and teacher.

A small number of Salmonella enterica serovars are host specific, only causing infection in one host species or a few closely related species. The diseases caused by broad-host-range and host-restricted Salmonella serovars also differ. Rearrangements can occur via recombination between short repeat sequences, but most large chromosomal rearrangements in bacteria occur by recombination between repeated sequences with several kbp of homology. IS200 is the most common IS element in Salmonella, it transposes infrequently. Thus, in contrast to many bacteria that have a high background of transposition-mediated rearrangements, most genome rearrangements in Salmonella are due to recombination. In contrast to the broad-host-range serovars, isolates of host-specific S. entericaserovars have large-scale chromosomal rearrangements resulting from recombination between the rrn operons. Inversions and translocations change the order of the chromosomal regions between the rrn operons from the conserved arrangement type found in the broad-host-range serovars to one of over fifty arrangement types identified so far in host-specific serovars. Comparative genomics has revealed that many host-specific pathogens show greater genome plasticity than closely related bacteria that live in a wider variety of environmental conditions. Although the genome rearrangements in other bacteria are often mediated by active transposons, it seems likely that the observed differences in genome plasticity are simply determined by the selective constraints of their distinct ecological niches. Genome rearrangements also have practical implications for foodborne pathogens, as they can complicate the identification and tracking of outbreak strains.

In this chapter, the author tells a story about the characterization of the operon from Salmonella. The author commenced his research trying to figure out why high phosphate concentrations inhibit cell division in Microcyclus flavus. In the final analysis, the most obvious change was in the distribution of phospholipids; however, it was not clear if this was the reason why cells stopped dividing or a secondary response to the inhibition of cell division. This led to his lifelong interest in membranes and convinced him that if he wanted to understand cause and effect, he will need to do genetics. His research in John’s lab focused on the regulation of proline utilization by the PutA protein, a bifunctional protein that could function as either a membrane-associated enzyme or a repressor. The proline utilization (put) operon allows cells to use proline as a sole source of carbon, nitrogen, and energy. In vitro studies confirmed that PutA binds to specific operator sites in the put control region. Instead of simply responding to the presence or absence of proline, induction of the put operon requires several physiological conditions needed for its catabolism: high concentrations of proline, an appropriate terminal electron acceptor, and the absence of other substrates that could compete for functional membrane binding sites.

One of the ways John Roth influenced many scientists was through teaching the Cold Spring Harbor Advanced Bacterial Genetics course. The course had a long tradition, evolving from earlier phage courses but retaining the core teaching approaches. Since its inception, the Phage course and subsequently the Advanced Bacterial Genetics course have trained many of the leaders in these fields. The molecular dissection of phage provided many important clues into central biological processes, but there were some questions that could not be answered with phage-questions that required studying cells directly. In 1950 Milislav Demerec began an offshoot of the Phage course that emphasized bacterial genetics. When both were consecutively taught, the Bacterial Genetics course immediately followed the Phage course, and many students took the two courses sequentially to obtain training in both phage and bacterial genetics. Over time many of the concepts and techniques from the Phage course were integrated into the Bacterial Genetics course until the two courses merged into a single course in 1971. A lab manual was published, which emphasized how genetics can be used to study pathogenic bacteria, a growing field with a dramatic need for researchers trained in bacterial genetics. Genetic characterization of diverse bacteria is important because they have practical applications and because the diversity of the organisms is likely to yield exciting new biological insights.

Salmonella enterica serovar Enteritidis is a leading cause of food poisoning in the USA and Europe. Although Salmonella serovars share many fimbrial operons, a few fimbriae are limited to specific Samonella serovars. SEF14 fimbriae are restricted to group D Salmonella and the genes encoding this virulence factor were acquired relatively recently. Genomic, genetic and gene expression studies have been integrated to investigate the ancestry, regulation and expression of the sef genes. Genomic comparisons of the Salmonella serovars sequenced revealed that the sef operon is inserted in leuX in Salmonella Enteritidis, Salmonella Paratyphi and Salmonella Typhi, and revealed the presence of a previously unidentified 25 kb pathogenicity island in Salmonella Typhimurium at this location. Salmonella Enteritidis contains a region of homology between the Salmonella virulence plasmid and the chromosome downstream of the sef operon. The sef operon itself consists of four co-transcribed genes, sefABCD, and adjacent to sefD there is an AraC-like transcriptional activator that is required for expression of the sef genes. Expression of the sef genes was optimal during growth in late exponential phase and was repressed during stationary phase. The regulation was coordinated by the RpoS sigma factor.

A PCR method was developed by which to rapidly and accurately determine the rrn arrangement of Salmonella enterica serovars. Primers were designed to the genomic regions flanking each of the seven rrn operons . PCR analysis using combinations of these primers will distinguish each of the possible arrangements of the rrn skeleton.

A fitness cost due to imbalanced replichores has been proposed to provoke chromosome rearrangements in Salmonella enterica serovars. To determine the impact of replichore imbalance on fitness, the relative fitness of isogenic Salmonella strains containing transposon-held duplications of various sizes and at various chromosomal locations was determined. Although duplication of certain genes influenced fitness, a replichore imbalance of up to 16° did not affect fitness.

Enteric bacteria possess multiple fimbriae, many of which play critical roles in attachment to epithelial cell surfaces. SEF14 fimbriae are only found in Salmonella enterica serovar Enteritidis ( S. enteritidis ) and closely related serovars, suggesting that SEF14 fimbriae may affect serovar-specific virulence traits. Despite evidence that SEF14 fimbriae are expressed by S. enteritidis in vivo , previous studies showed that SEF14 fimbriae do not mediate adhesion to the intestinal epithelium. Therefore, we tested whether SEF14 fimbriae are required for virulence at a stage in infection after the bacteria have passed the intestinal barrier. Polar mutations that disrupt the entire sef operon decreased virulence in mice more than 1,000-fold. Nonpolar mutations that disrupted sefA (encoding the major structural subunit) did not affect virulence, but mutations that disrupted sefD (encoding the putative adhesion subunit) resulted in a severe virulence defect. The results indicate that the putative SEF14 adhesion subunit is specifically required for a stage of the infection subsequent to transit across the intestinal barrier. Therefore, we tested whether SefD is required for uptake or survival in macrophages. The majority of wild-type bacteria were detected inside macrophages soon after i.p. infection, but the sefD mutants were not readily internalized by peritoneal macrophages. These results indicate that the potential SEF14 adhesion subunit is essential for efficient uptake or survival of S. enteritidis in macrophages. This report describes a role of fimbriae in intracellular infection, and indicates that fimbriae may be required for systemic infections at stages beyond the initial colonization of host epithelial surfaces.

Experimental dissection of bacterial genomes requires a well-developed set of genetic tools, but many bacteria lack the essential tools required for genetic analysis. Recombination of a region of chromosomal DNA from poorly characterized donor bacteria with the chromosome of a suitable surrogate host creates a genetically malleable hybrid, providing a short-cut for the detailed genetic analysis of the substituted genes. However, recombination between closely related but nonidentical DNA sequences ("homeologous recombination") is strongly inhibited, posing a powerful barrier to gene exchange between bacteria and a major impediment to the construction of genetic hybrids. By taking advantage of mutS and recD mutant recipients, it is possible to effectively overcome the recombination barrier, allowing construction of genetic hybrids in a related surrogate host. Once stably recombined into the recipient chromosome, the donor DNA can be studied with all the genetic tools available in the surrogate host. In addition to facilitating standard genetic analysis, use of a surrogate host can provide novel approaches to study the physiological roles of unique genes from poorly characterized bacteria.

The Genetics Society of America annually honors members who have made outstanding contributions to genetics. The Thomas Hunt Morgan Medal recognizes a lifetime contribution to the science of genetics. The Genetics Society of America Medal recognizes particularly outstanding contributions to the

PutA is a multifunctional, peripheral membrane protein which functions both as an autogenous transcriptional repressor and the enzyme which catalyzes the two-step conversion of proline to glutamate in Salmonella typhimurium and Escherichia coli. To understand how PutA associates with the membrane, we determined the role of FAD redox and membrane components in PutA–membrane association. Reduction of the tightly bound FAD is required for both derepression of the put operon and membrane association of PutA. FADH2 alters the conformation of PutA, resulting in an increased hydrophobicity. Previous studies used enzymatic activity as an assay for membrane association and concluded that electron transfer from the reduced FAD in PutA to the membrane is required for the PutA–membrane interaction. However, direct physical assays of PutA association with membrane vesicles from quinone deficient mutants demonstrated that although electron transfer is essential for proline dehydrogenase activity, it is not required for PutA–membrane association per se. Furthermore, PutA efficiently associated with liposomes, indicating that PutA–membrane association does not require interactions with other membrane proteins. PutA enzymatic activity can be efficiently reconstituted with liposomes containing ubiquinone and cytochrome bo, confirming that proline dehydrogenase can pass electrons directly to the quinone pool. These results indicate that PutA–membrane association is due strictly to a protein–lipid interaction initiated by reduction of FAD.

E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

When the Rockefeller Institute for Medical Research was founded in 1901, infectious diseases were the most serious threat to human health—typhoid fever, tuberculosis, and diphtheria killed many people around the world, primarily poorer people but not sparing the affluent. In fact, the death of John Rockefeller's grandson of scarlet fever that year is said to have been the stimulus for the creation of the Rockefeller Institute.

The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli . We used P22 challenge phage carrying the put control region from K. aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo. Mutations in an asymmetric 30-bp region prevented DNA binding by NAC. Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium , which is not regulated by NAC.

Summary: Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy clone of the proY + gene or both a single copy of the proY + gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.