Transcriptomic analysis of the Myxococcus xanthus FruA regulon, and comparative developmental transcriptomic analysis of two fruiting body forming species, Myxococcus xanthus and Myxococcus stipitatus.

BACKGROUND: The Myxococcales are well known for their predatory and developmental social processes, and for the molecular complexity of regulation of these processes. Many species within this order have unusually large genomes compared to other bacteria, and their genomes have many genes that are unique to one specific sequenced species or strain. Here, we describe RNAseq based transcriptome analysis of the FruA regulon of Myxococcus xanthus and a comparative RNAseq analysis of two Myxococcus species, M. xanthus and Myxococcus stipitatus, as they respond to starvation and begin forming fruiting bodies. RESULTS: We show that both species have large numbers of genes that are developmentally regulated, with over half the genome showing statistically significant changes in expression during development in each species. We also included a non-fruiting mutant of M. xanthus that is missing the transcriptional regulator FruA to identify the direct and indirect FruA regulon and to identify transcriptional changes that are specific to fruiting and not just the starvation response. We then identified Interpro gene ontologies and COG annotations that are significantly up- or down-regulated during development in each species. Our analyses support previous data for M. xanthus showing developmental upregulation of signal transduction genes, and downregulation of genes related to cell-cycle, translation, metabolism, and in some cases, DNA replication. Gene expression in M. stipitatus follows similar trends. Although not all specific genes show similar regulation patterns in both species, many critical developmental genes in M. xanthus have conserved expression patterns in M. stipitatus, and some groups of otherwise unstudied orthologous genes share expression patterns. CONCLUSIONS: By identifying the FruA regulon and identifying genes that are similarly and uniquely regulated in two different species, this work provides a more complete picture of transcription during Myxococcus development. We also provide an R script to allow other scientists to mine our data for genes whose expression patterns match a user-selected gene of interest.

MglC, a Paralog of Myxococcus xanthus GTPase-Activating Protein MglB, Plays a Divergent Role in Motility Regulation.

UNLABELLED: In order to optimize interactions with their environment and one another, bacteria regulate their motility. In the case of the rod-shaped cells of Myxococcus xanthus, regulated motility is essential for social behaviors. M. xanthus moves over surfaces using type IV pilus-dependent motility and gliding motility. These two motility systems are coordinated by a protein module that controls cell polarity and consists of three polarly localized proteins, the small G protein MglA, the cognate MglA GTPase-activating protein MglB, and the response regulator RomR. Cellular reversals are induced by the Frz chemosensory system, and the output response regulator of this system, FrzZ, interfaces with the MglA/MglB/RomR module to invert cell polarity. Using a computational approach, we identify a paralog of MglB, MXAN_5770 (MglC). Genetic epistasis experiments demonstrate that MglC functions in the same pathway as MglA, MglB, RomR, and FrzZ and is important for regulating cellular reversals. Like MglB, MglC localizes to the cell poles asymmetrically and with a large cluster at the lagging pole. Correct polar localization of MglC depends on RomR and MglB. Consistently, MglC interacts directly with MglB and the C-terminal output domain of RomR, and we identified a surface of MglC that is necessary for the interaction with MglB and for MglC function. Together, our findings identify an additional member of the M. xanthus polarity module involved in regulating motility and demonstrate how gene duplication followed by functional divergence can add a layer of control to the complex cellular processes of motility and motility regulation. IMPORTANCE: Gene duplication and the subsequent divergence of the duplicated genes are important evolutionary mechanisms for increasing both biological complexity and regulation of biological processes. The bacterium Myxococcus xanthus is a soil bacterium with an unusually large genome that carries out several social processes, including predation of other bacterial species and formation of multicellular, spore-filled fruiting bodies. One feature of the large M. xanthus genome is that it contains many gene duplications. Here, we compare the products of one example of gene duplication and divergence, in which a paralog of the cognate MglA GTPase-activating protein MglB has acquired a different and opposing role in the regulation of cellular polarity and motility, processes critical to the bacterium's social behaviors.

The lineage-specific gene ponzr1 is essential for zebrafish pronephric and pharyngeal arch development.

The Homeobox (Hox) and Paired box (Pax) gene families are key determinants of animal body plans and organ structure. In particular, they function within regulatory networks that control organogenesis. How these conserved genes elicit differences in organ form and function in response to evolutionary pressures is incompletely understood. We molecularly and functionally characterized one member of an evolutionarily dynamic gene family, plac8 onzin related protein 1 (ponzr1), in the zebrafish. ponzr1 mRNA is expressed early in the developing kidney and pharyngeal arches. Using ponzr1-targeting morpholinos, we show that ponzr1 is required for formation of the glomerulus. Loss of ponzr1 results in a nonfunctional glomerulus but retention of a functional pronephros, an arrangement similar to the aglomerular kidneys found in a subset of marine fish. ponzr1 is integrated into the pax2a pathway, with ponzr1 expression requiring pax2a gene function, and proper pax2a expression requiring normal ponzr1 expression. In addition to pronephric function, ponzr1 is required for pharyngeal arch formation. We functionally demonstrate that ponzr1 can act as a transcription factor or co-factor, providing the first molecular mode of action for this newly described gene family. Together, this work provides experimental evidence of an additional mechanism that incorporates evolutionarily dynamic, lineage-specific gene families into conserved regulatory gene networks to create functional organ diversity.

SinR is a mutational target for fine-tuning biofilm formation in laboratory-evolved strains of Bacillus subtilis.

BACKGROUND: Bacteria often form multicellular, organized communities known as biofilms, which protect cells from a variety of environmental stresses. During biofilm formation, bacteria secrete a species-specific matrix; in Bacillus subtilis biofilms, the matrix consists of protein polymers and exopolysaccharide. Many domesticated strains of B. subtilis have a reduced ability to form biofilms, and we conducted a two-month evolution experiment to test whether laboratory culturing provides selective pressure against biofilm formation in B. subtilis. RESULTS: Bacteria grown in two-month-long batch culture rapidly diversified their biofilm-forming characteristics, exhibiting highly diverse colony morphologies on LB plates in the initial ten days of culture. Generally, this diversity decreased over time; however, multiple types of colony morphology remained in our final two-month-old populations, both under shaking and static conditions. Notably, while our final populations featured cells that produce less biofilm matrix than did the ancestor, cells overproducing biofilm matrix were present as well. We took a candidate-gene approach to identify mutations in the strains that overproduced matrix and found point mutations in the biofilm-regulatory gene sinR. Introducing these mutations into the ancestral strain phenocopied or partially phenocopied the evolved biofilm phenotypes. CONCLUSIONS: Our data suggest that standard laboratory culturing conditions do not rapidly select against biofilm formation. Although biofilm matrix production is often reduced in domesticated bacterial strains, we found that matrix production may still have a fitness benefit in the laboratory. We suggest that adaptive specialization of biofilm-forming species can occur through mutations that modulate biofilm formation as in B. subtilis.

Five draft genome assemblies from Bacillaceae isolated from a degraded wetland environment.

We isolated five Bacillaceae from a degraded wetland environment and sequenced their genomes using Illumina NextSeq. Here, we report draft genome sequences of Bacillus velezensus-SC119, Priestia megaterium strain SC120, Bacillus zhangzhouensis strain SC123, Bacillus pumilis strain SC124, and Bacillus idriensis strain SC127. The genomes range between 3,657,353 and 5,772,725 bp with % GC between 37.62% and 46.38%.

Osmotic pressure can regulate matrix gene expression in Bacillus subtilis.

Many bacteria organize themselves into structurally complex communities known as biofilms in which the cells are held together by an extracellular matrix. In general, the amount of extracellular matrix is related to the robustness of the biofilm. Yet, the specific signals that regulate the synthesis of matrix remain poorly understood. Here we show that the matrix itself can be a cue that regulates the expression of the genes involved in matrix synthesis in Bacillus subtilis. The presence of the exopolysaccharide component of the matrix causes an increase in osmotic pressure that leads to an inhibition of matrix gene expression. We further show that non-specific changes in osmotic pressure also inhibit matrix gene expression and do so by activating the histidine kinase KinD. KinD, in turn, directs the phosphorylation of the master regulatory protein Spo0A, which at high levels represses matrix gene expression. Sensing a physical cue such as osmotic pressure, in addition to chemical cues, could be a strategy to non-specifically co-ordinate the behaviour of cells in communities composed of many different species.

Draft Genome Sequences for 6 Isolates of Endospore-Forming Class Bacilli Species Isolated from Soil from a Suburban, Wooded, Developed Space.

Here, we report draft genome sequences of Bacillus pseudomycoides SC107, Rossellomorea sp. SC111, Peribacillus frigoritolerans SC112, Priestia megaterium SC114, Paenibacillus sp. SC116, and Lysinibacillus fusiformis SC117, isolated from a campus wooded area in Loudonville, NY. Genomes were sequenced using the Illumina NextSeq system. The genomes range between 4,381,526 and 6,065,181 bp with GC contents between 35.33% and 43.46%.

Tracing the domestication of a biofilm-forming bacterium.

Over the course of more than a century of laboratory experimentation, Bacillus subtilis has become "domesticated," losing its ability to carry out many behaviors characteristic of its wild ancestors. One such characteristic is the ability to form architecturally complex communities, referred to as biofilms. Previous work has shown that the laboratory strain 168 forms markedly attenuated biofilms compared with the wild strain NCIB3610 (3610), even after repair of a mutation in sfp (a gene involved in surfactin production) previously known to impair biofilm formation. Here, we show that in addition to the sfp mutation, mutations in epsC, swrA, and degQ are necessary and sufficient to explain the inability of the laboratory strain to produce robust biofilms. Finally, we show that the architecture of the biofilm is markedly influenced by a large plasmid present in 3610 but not 168 and that the effect of the plasmid can be attributed to a gene we designate rapP. When rapP is introduced into 168 together with wild-type alleles of sfp, epsC, swrA, and degQ, the resulting repaired laboratory strain forms biofilms that are as robust as and essentially indistinguishable in architecture from those of the wild strain, 3610. Thus, domestication of B. subtilis involved the accumulation of four mutations and the loss of a plasmid-borne gene.

Spatial regulation of histidine kinases governing biofilm formation in Bacillus subtilis.

Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community.

An Adaptable and Modular Set of Laboratory Exercises Connecting Genotype to Phenotype in Sporulating Bacillus subtilis.

Practical lab exercises that help students draw connections between genotype and phenotype, and make and test predictions about the identity of mutants, are invaluable in college-level cell biology, genetics, and microbiology courses. While many bacteria are easy to grow and manipulate within the time and resource constraints of a laboratory course, their phenotypes are not always observable or relevant-seeming to college students. Here, we leverage sporulation by the bacterium Bacillus subtilis, a well-characterized and genetically tractable system, to create 5 adaptable lab exercises that can be implemented in different combinations to suit the needs of a variety of courses and instruction modes. Because phenotypic changes during sporulation are striking morphological changes to cells that are easily observable with basic light microscopy, and because spore-forming bacteria related to B. subtilis have clear applications for human and environmental health, these exercises have the potential to engage students' interest while introducing and reinforcing key concepts in microbiology, cell biology, and genetics.

A novel regulatory protein governing biofilm formation in Bacillus subtilis.

Production of an extracellular matrix is a hallmark of biofilm formation. In the spore-forming bacterium Bacillus subtilis, the matrix consists of an exopolysaccharide, which is specified by the epsA-O operon, and a secreted protein TasA, which is encoded by the yqxM-sipW-tasA operon. Past and present evidence establish that the epsA-O and yqxM-sipW-tasA operons are controlled by the repressor proteins SinR and AbrB. Here, we report the identification of a novel regulatory protein Slr that promotes transcription of the yqxM-sipW-tasA operon but is not needed for expression of the epsA-O operon. We further show that the gene for Slr is itself under the negative control of SinR and AbrB. These findings reveal that matrix production is governed by an intricate network involving the interplay of negatively and positively acting regulatory proteins.

Crosslinked, Glassy Styrenic Surfactants Stabilize Quantum Dots Against Environmental Extremes.

Semiconductor, quantum dot (QD) nanoparticles (including CdSe/ZnS, CdTe/ZnS, and CdSe) were encapsulated within cross-linked shells of amphiphilic polystyrene-block-poly(acrylic acid) block copolymer. Transmission electron microscopy revealed that each particle was surrounded by a uniform, layer of copolymer, and that the average diameter of the resulting QD-core micelles was between 25 and 50 nm, depending on the conditions of particle assembly. Overall, we found that aqueous suspensions of these QDs were substantially more stable to heat and pH than particles with other surface preparations; we argue that the enhanced stability is due to the uniform, hydrophobic coating of polystyrene around each particle and the reinforcement of this layer by shell-cross-linking. The biocompatibility of these particles was investigated by microinjection of particle suspension into live zebrafish embryos. The particles permanently stained the fish vasculature, but did not interfere with the normal development of the fish. We propose that QDs encapsulated in cross-linked block-copolymer shells allow QDs to be used in biological or biotechnological protocols requiring harsh reaction conditions.

Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila.

Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. Here we show that neural activity directs the mRNA of the Drosophila Ca(2+), Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. We show that mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, we propose that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory.