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1.  Sequential evolution of bacterial morphology by co-option of a developmental regulator 
Nature  2014;506(7489):489-493.
What mechanisms underlie the transitions responsible for the diverse shapes observed in the living world? While bacteria display a myriad of morphologies1, the mechanisms responsible for the evolution of bacterial cell shape are not understood. We investigated morphological diversity in a group of bacteria that synthesize an appendage-like extension of the cell envelope called the stalk2,3. The location and number of stalks varies among species, as exemplified by three distinct sub-cellular positions of stalks within a rod-shaped cell body: polar in the Caulobacter genus, and sub-polar or bi-lateral in the Asticcacaulis genus4. Here we show that a developmental regulator of Caulobacter crescentus, SpmX5, was co-opted in the Asticcacaulis genus to specify stalk synthesis at either the sub-polar or bi-lateral positions. We show that stepwise evolution of a specific region of SpmX led to the gain of a new function and localization of this protein, which drove the sequential transition in stalk positioning. Our results indicate that evolution of protein function, co-option, and modularity are key elements in the evolution of bacterial morphology. Therefore, similar evolutionary principles of morphological transitions apply to both single-celled prokaryotes and multicellular eukaryotes.
PMCID: PMC4035126  PMID: 24463524
2.  Biological Consequences and Advantages of Asymmetric Bacterial Growth 
Annual review of microbiology  2013;67:417-435.
Asymmetries in cell growth and division occur in eukaryotes and prokaryotes alike. Even seemingly simple and morphologically symmetric cell division processes belie inherent underlying asymmetries in the composition of the resulting daughter cells. We consider the types of asymmetry that arise in various bacterial cell growth and division processes, which include both conditionally activated mechanisms and constitutive, hardwired aspects of bacterial life histories. Although asymmetry disposes some cells to the deleterious effects of aging, it may also benefit populations by efficiently purging accumulated damage and rejuvenating newborn cells. Asymmetries may also generate phenotypic variation required for successful exploitation of variable environments, even when extrinsic changes outpace the capacity of cells to sense and respond to challenges. We propose specific experimental approaches to further develop our understanding of the prevalence and the ultimate importance of asymmetric bacterial growth.
PMCID: PMC4001765  PMID: 23808335
asymmetry; peptidoglycan; sporulation; aging; damage
3.  The adhesive and cohesive properties of a bacterial polysaccharide adhesin are modulated by a deacetylase 
Molecular microbiology  2013;88(3):486-500.
△Bacterial exopolysaccharide synthesis is a prevalent and indispensible activity in many biological processes, including surface adhesion and biofilm formation. In Caulobacter crescentus, surface attachment and subsequent biofilm growth depend on the ability to synthesize an adhesive polar polysaccharide known as the holdfast. In this work, we show that polar polysaccharide synthesis is a conserved phenomenon among Alphaproteobacterial species closely related to C. crescentus. Among them, mutagenesis of Asticcacaulis biprosthecum showed that disruption of the hfsH gene, which encodes a putative polysaccharide deacetylase, leads to accumulation of holdfast in the culture supernatant. Examination of the hfsH deletion mutant in C. crescentus revealed that this strain synthesizes holdfast; however like the A. biprosthecum hfsH mutant, the holdfasts are shed into the medium and have decreased adhesiveness and cohesiveness. Site-directed mutagenesis at the predicted catalytic site of C. crescentus HfsH phenocopied the ΔhfsH mutant and abolished the esterase activity of HfsH. In contrast, overexpression of HfsH increased cell adherence without increasing holdfast synthesis. We conclude that the polysaccharide deacetylase activity of HfsH is required for the adhesive and cohesive properties of the holdfast, as well as for the anchoring of the holdfast to the cell envelope.
PMCID: PMC3633684  PMID: 23517529
4.  In situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with Fluorescent D-Amino Acids.** 
Tracking a bug’s life: We describe the first direct and universal approach for labeling peptidoglycan (PG) of diverse bacteria by exploiting the surprising tolerance of cells for incorporating unnatural D-amino acids of various sizes and functionalities. These non-toxic D-amino acids preferably label the sites of active PG synthesis, enabling fine spatiotemporal tracking of cell wall dynamics in phylogenetically and morphologically diverse bacteria.
PMCID: PMC3589519  PMID: 23055266
bacteria; biosensors; D-amino acids; fluorescent probes; peptidoglycan
5.  Microfluidic Device for Automated Synchronization of Bacterial Cells 
Analytical chemistry  2012;84(20):8571-8578.
We report the development of an automated microfluidic “baby machine” to synchronize the bacterium Caulobacter crescentus on-chip and to move the synchronized populations downstream for analysis. The microfluidic device is fabricated from three-layers of poly(dimethylsiloxane) and has integrated pumps and valves to control the movement of cells and media. This synchronization method decreases incubation time and media consumption and improves synchrony quality compared to the conventional plate-release technique. Synchronized populations are collected from the device at intervals as short as 10 min and at any time over four days. Flow cytometry and fluorescence cell tracking are used to determine synchrony quality, and cell populations synchronized in M2G and PYE media contain >70% and >80% swarmer cells, respectively. Our on-chip method overcomes limitations with conventional physical separation methods that consume large volumes of media, require manual manipulations, have lengthy incubation times, are limited to one collection, and lack precise temporal control of collection times.
PMCID: PMC3484264  PMID: 23030473
6.  GeneclusterViz: a tool for conserved gene cluster visualization, exploration and analysis 
Bioinformatics  2012;28(11):1527-1529.
Motivation: Gene clusters are arrangements of functionally related genes on a chromosome. In bacteria, it is expected that evolutionary pressures would conserve these arrangements due to the functional advantages they provide. Visualization of conserved gene clusters across multiple genomes provides key insights into their evolutionary histories. Therefore, a software tool that enables visualization and functional analyses of gene clusters would be a great asset to the biological research community.
Results: We have developed GeneclusterViz, a Java-based tool that allows for the visualization, exploration and downstream analyses of conserved gene clusters across multiple genomes. GeneclusterViz combines an easy-to-use exploration interface for gene clusters with a host of other analysis features such as multiple sequence alignments, phylogenetic analyses and integration with the KEGG pathway database.
Supplementary information: Supplementary data are available at Bioinformatics online.
PMCID: PMC3356842  PMID: 22495752
7.  Surface contact stimulates the just-in-time deployment of bacterial adhesins 
Molecular microbiology  2011;83(1):41-51.
The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive.
PMCID: PMC3245333  PMID: 22053824
8.  Children With Co-Occurring Anxiety and Externalizing Disorders: Family Risks and Implications for Competence 
This study used data from 340 mother–child dyads to examine characteristics of children with co-occurring diagnoses of anxiety and externalizing disorders and compared them with children with a sole diagnosis or no diagnosis. Comparisons were made using 4 child-diagnostic groups: anxiety-only, externalizing-only, co-occurrence, and no-problem groups. Most mothers were characterized by low income and histories of psychiatric diagnoses during the child’s lifetime. Analyses using multinomial logistic regressions found the incidence of co-occurring childhood disorders to be significantly linked with maternal affective/anxiety disorders during the child’s lifetime. In exploring implications for developmental competence, we found the co-occurrence group to have the lowest level of adaptive functioning among the 4 groups, faring significantly worse than the no-problem group on both academic achievement and intelligence as assessed by standardized tests. Findings underscore the importance of considering co-occurring behavior problems as a distinct phenomenon when examining children’s developmental outcomes.
PMCID: PMC3490217  PMID: 20099944
co-occurring psychiatric disorders; anxiety disorders; externalizing disorders
9.  Conceptual Issues in Studies of Resilience 
We begin this article by considering the following critical conceptual issues in research on resilience: (1) distinctions between protective, promotive, and vulnerability factors; (2) the need to unpack underlying processes; (3) the benefits of within-group experimental designs; and (4) the advantages and potential pitfalls of an overwhelming scientific focus on biological and genetic factors (to the relative exclusion of familial and contextual ones). The next section of the article is focused on guidelines for the selection of vulnerability and protective processes in future research. From a basic science standpoint, it is useful and appropriate to investigate all types of processes that might significantly affect adjustment among at-risk individuals. If the research is fundamentally applied in nature, however, it would be most expedient to focus on risk modifiers that have high potential to alter individuals’ overall life circumstances. The final section of this article considers conceptual differences between contemporary resilience research on children versus adults. Issues include differences in the types and breadth of outcomes (e.g., the tendencies to focus on others’ ratings of competence among children and on self-reports of well-being among adults respectively).
PMCID: PMC3480733  PMID: 17347344
resilience; protective processes; risk modifiers; interventions
10.  Polarity and the diversity of growth mechanisms in bacteria 
Bacterial cell growth is a complex process consisting of two distinct phases: cell elongation and septum formation prior to cell division. Although bacteria have evolved several different mechanisms for cell growth, it is clear that tight spatial and temporal regulation of peptidoglycan synthesis is a common theme. In this review, we discuss bacterial cell growth with a particular emphasis on bacteria that utilize tip extension as a mechanism for cell elongation. We describe polar growth among diverse bacteria and consider the advantages and consequences of this mode of cell elongation.
PMCID: PMC3193591  PMID: 21736947
cell elongation; polar growth; DivIVA; peptidoglycan
11.  Genome Sequences of Eight Morphologically Diverse Alphaproteobacteria▿  
Journal of Bacteriology  2011;193(17):4567-4568.
The Alphaproteobacteriacomprise morphologically diverse bacteria, including many species of stalked bacteria. Here we announce the genome sequences of eight alphaproteobacteria, including the first genome sequences of species belonging to the genera Asticcacaulis, Hirschia, Hyphomicrobium, and Rhodomicrobium.
PMCID: PMC3165538  PMID: 21705585
12.  Complete genome sequence of Hirschia baltica type strain (IFAM 1418T) 
Standards in Genomic Sciences  2011;5(3):287-297.
The family Hyphomonadaceae within the Alphaproteobacteria is largely comprised of bacteria isolated from marine environments with striking morphologies and an unusual mode of cell growth. Here, we report the complete genome sequence Hirschia baltica, which is only the second a member of the Hyphomonadaceae with a published genome sequence. H. baltica is of special interest because it has a dimorphic life cycle and is a stalked, budding bacterium. The 3,455,622 bp long chromosome and 84,492 bp plasmid with a total of 3,222 protein-coding and 44 RNA genes were sequenced as part of the DOE Joint Genome Institute Program CSP 2008.
PMCID: PMC3368421  PMID: 22675580
aerobic; chemoheterotrophic; mesophile; Gram-negative; motile; budding; stalk-forming; Hyphomonadaceae; Alphaproteobacteria; CSP 2008
13.  Microchannel-Nanopore Device for Bacterial Chemotaxis Assays 
Analytical chemistry  2010;82(22):9357-9364.
Motile bacteria bias the random walk of their motion in response to chemical gradients by the process termed chemotaxis, which allows cells to accumulate in favorable environments and disperse from less favorable ones. In this work, we describe a simple microchannel-nanopore device that establishes a stable chemical gradient for chemotaxis assays in ≤ 1 min. Chemoattractant is dispensed by diffusion through 10 nm diameter pores at the intersection of two microchannels. This design requires no external pump and minimizes the effect of transmembrane pressure, resulting in a stable, reproducible gradient. The microfluidic platform facilitates microscopic observation of individual cell trajectories, and chemotaxis is quantified by monitoring changes in cell swimming behavior in the vicinity of the intersection. We validate this system by measuring the chemotactic response of an aquatic bacterium, Caulobacter crescentus, to xylose concentrations from 1.3 μM to 1.3 M. Additionally, we make an unanticipated observation of increased turn frequency in a chemotaxis-impaired mutant which provides new insight into the chemotaxis pathway in C. crescentus.
PMCID: PMC2992552  PMID: 20961116
14.  A localized multimeric anchor attaches the Caulobacter holdfast to the cell pole 
Molecular microbiology  2010;76(2):409-427.
Caulobacter crescentus attachment is mediated by the holdfast, a complex of polysaccharide anchored to the cell by HfaA, HfaB and HfaD. We show that all three proteins are surface-exposed outer membrane (OM) proteins. HfaA is similar to fimbrial proteins and assembles into a high molecular weight (HMW) form requiring HfaD, but not holdfast polysaccharide. The HfaD HMW form is dependent on HfaA but not on holdfast polysaccharide. We show that HfaA and HfaD form homomultimers and that they require HfaB for stability and OM translocation. All three proteins localize to the late predivisional flagellar pole, remain at this pole in swarmer cells, and localize at the stalk tip after the stalk is synthesized at the same pole. Hfa protein localization requires the holdfast polysaccharide secretion proteins and the polar localization factor PodJ. A hfaB mutant is much more severely deficient in adherence and holdfast attachment than hfaA and hfaD mutants. A hfaA, hfaD double mutant phenocopies either single mutant, suggesting that HfaB is involved in holdfast attachment beyond secretion of HfaA and HfaD. We hypothesize HfaB secretes HfaA and HfaD across the outer membrane, and the three proteins form a complex anchoring the holdfast to the stalk.
PMCID: PMC2908716  PMID: 20233308
attachment; Caulobacter crescentus; Hfa; holdfast
15.  Complex regulatory pathways coordinate cell cycle progression and development in Caulobacter crescentus 
Caulobacter crescentus has become the predominant bacterial model system to study the regulation of cell cycle progression. Stage specific processes such as chromosome replication and segregation, and cell division are coordinated with the development of four polar structures: the flagellum, pili, stalk, and holdfast. The production, activation, localization, and proteolysis of specific regulatory proteins at precise times during the cell cycle culminate in the ability of the cell to produce two physiologically distinct daughter cells. We examine the recent advances that have enhanced our understanding of the mechanisms of temporal and spatial regulation that occur during cell cycle progression.
PMCID: PMC2621326  PMID: 18929067
16.  Extracurricular Involvement Among Affluent Youth: A Scapegoat for “Ubiquitous Achievement Pressures”? 
Developmental psychology  2006;42(3):583-597.
It has been suggested that overscheduling of upper-class youth might underlie the high distress and substance use documented among them. This assumption was tested by considering suburban 8th graders’ involvement in different activities along with their perceptions of parental attitudes toward achievement. Results indicated negligible evidence for deleterious effects of high extracurricular involvement per se. Far more strongly implicated was perceived parent criticism for both girls and boys as well as the absence of after-school supervision. Low parent expectations connoted significant vulnerability especially for boys. The findings indicate that at least among early adolescents, converging scientific and media reports may have scapegoated extracurricular involvements, to some degree, as an index of ubiquitous achievement pressures in affluent communities.
PMCID: PMC1852438  PMID: 16756447
affluence; extracurricular activities; achievement pressure; parent criticism; parent expectations
17.  Maximizing resilience through diverse levels of inquiry: Prevailing paradigms, possibilities, and priorities for the future 
Development and psychopathology  2007;19(3):931-955.
The study of resilience has two core characteristics: it is fundamentally applied in nature, seeking to use scientific knowledge to maximize well-being among those at risk, and it draws on expertise from diverse scientific disciplines. Recent advances in biological processes have confirmed the profound deleterious effects of harsh caregiving environments, thereby underscoring the importance of early interventions. What remains to be established at this time is the degree to which insights on particular biological processes (e.g., involving specific brain regions, genes, or hormones) will be applied in the near future to achieve substantial reductions in mental health disparities. Aside from biology, resilience developmental researchers would do well to draw upon relevant evidence from other behavioral sciences as well, notably anthropology as well as family, counseling, and social psychology. Scientists working with adults and with children must remain vigilant to the advances and missteps in each others' work, always ensuring caution in conveying messages about the “innateness” of resilience or its prevalence across different subgroups. Our future research agenda must prioritize reducing abuse and neglect in close relationships; deriving the “critical ingredients” in effective interventions and going to scale with these; working collaboratively to refine theory on the construct; and responsibly, proactively disseminating what we have learned about the nature, limits, and antecedents of resilient adaptation across diverse at-risk groups.
PMCID: PMC2190297  PMID: 17705909

Results 1-17 (17)