Lattice gas cellular automata model for rippling and aggregation in myxobacteria

A lattice-gas cellular automaton (LGCA) model is used to simulate rippling and aggregation in myxobacteria. An efficient way of representing cells of different cell size, shape and orientation is presented that may be easily extended to model later stages of fruiting body formation. This LGCA model is designed to investigate whether a refractory period, a minimum response time, a maximum oscillation period and non-linear dependence of reversals of cells on C-factor are necessary assumptions for rippling. It is shown that a refractory period of 2-3 minutes, a minimum response time of up to 1 minute and no maximum oscillation period best reproduce rippling in the experiments of {\it Myxoccoccus xanthus}. Non-linear dependence of reversals on C-factor is critical at high cell density. Quantitative simulations demonstrate that the increase in wavelength of ripples when a culture is diluted with non-signaling cells can be explained entirely by the decreased density of C-signaling cells. This result further supports the hypothesis that levels of C-signaling quantitatively depend on and modulate cell density. Analysis of the interpenetrating high density waves shows the presence of a phase shift analogous to the phase shift of interpenetrating solitons. Finally, a model for swarming, aggregation and early fruiting body formation is presented

Identification and Localization of Myxococcus xanthus Porins and Lipoproteins

Myxococcus xanthus DK1622 contains inner (IM) and outer membranes (OM) separated by a peptidoglycan layer. Integral membrane, β-barrel proteins are found exclusively in the OM where they form pores allowing the passage of nutrients, waste products and signals. One porin, Oar, is required for intercellular communication of the C-signal. An oar mutant produces CsgA but is unable to ripple or stimulate csgA mutants to develop suggesting that it is the channel for C-signaling. Six prediction programs were evaluated for their ability to identify β-barrel proteins. No program was reliable unless the predicted proteins were first parsed using Signal P, Lipo P and TMHMM, after which TMBETA-SVM and TMBETADISC-RBF identified β-barrel proteins most accurately. 228 β-barrel proteins were predicted from among 7331 protein coding regions, representing 3.1% of total genes. Sucrose density gradients were used to separate vegetative cell IM and OM fractions, and LC-MS/MS of OM proteins identified 54 β-barrel proteins. Another class of membrane proteins, the lipoproteins, are anchored in the membrane via a lipid moiety at the N-terminus. 44 OM proteins identified by LC-MS/MS were predicted lipoproteins. Lipoproteins are distributed between the IM, OM and ECM according to an N-terminal sorting sequence that varies among species. Sequence analysis revealed conservation of alanine at the +7 position of mature ECM lipoproteins, lysine at the +2 position of IM lipoproteins, and no noticable conservation within the OM lipoproteins. Site directed mutagenesis and immuno transmission electron microscopy showed that alanine at the +7 position is essential for sorting of the lipoprotein FibA into the ECM. FibA appears at normal levels in the ECM even when a +2 lysine is added to the signal sequence. These results suggest that ECM proteins have a unique method of secretion. It is now possible to target lipoproteins to specific IM, OM and ECM locations by manipulating the amino acid sequence near the +1 cysteine processing site

Hypertension in mice lacking 11beta-hydroxysteroid dehydrogenase type 2

Deficiency of 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) in humans leads to the syndrome of apparent mineralocorticoid excess (SAME), in which cortisol illicitly occupies mineralocorticoid receptors, causing sodium retention, hypokalemia, and hypertension. However, the disorder is usually incompletely corrected by suppression of cortisol, suggesting additional and irreversible changes, perhaps in the kidney. To examine this further, we produced mice with targeted disruption of the 11β-HSD2 gene. Homozygous mutant mice (11β-HSD2(–/–)) appear normal at birth, but ∼50% show motor weakness and die within 48 hours. Both male and female survivors are fertile but exhibit hypokalemia, hypotonic polyuria, and apparent mineralocorticoid activity of corticosterone. Young adult 11β-HSD2(–/–) mice are markedly hypertensive, with a mean arterial blood pressure of 146 ± 2 mmHg, compared with 121 ± 2 mmHg in wild-type controls and 114 ± 4 mmHg in heterozygotes. The epithelium of the distal tubule of the nephron shows striking hypertrophy and hyperplasia. These histological changes do not readily reverse with mineralocorticoid receptor antagonism in adulthood. Thus, 11β-HSD2(–/–) mice demonstrate the major features of SAME, providing a unique rodent model to study the molecular mechanisms of kidney resetting leading to hypertension. J. Clin. Invest. 103:683–689 (1999

A Novel Cold-Adapted Lipase from Sorangium cellulosum Strain So0157-2: Gene Cloning, Expression, and Enzymatic Characterization

Genome sequencing of cellulolytic myxobacterium Sorangium cellulosum reveals many open-reading frames (ORFs) encoding various degradation enzymes with low sequence similarity to those reported, but none of them has been characterized. In this paper, a predicted lipase gene (lipA) was cloned from S. cellulosum strain So0157-2 and characterized. lipA is 981-bp in size, encoding a polypeptide of 326 amino acids that contains the pentapeptide (GHSMG) and catalytic triad residues (Ser114, Asp250 and His284). Searching in the GenBank database shows that the LipA protein has only the 30% maximal identity to a human monoglyceride lipase. The novel lipA gene was expressed in Escherichia coli BL21 and the recombinant protein (r-LipA) was purified using Ni-NTA affinity chromatography. The enzyme hydrolyzed the p-nitrophenyl (pNP) esters of short or medium chain fatty acids (≤C10), and the maximal activity was on pNP acetate. The r- LipA is a cold-adapted lipase, with high enzymatic activity in a wide range of temperature and pH values. At 4 °C and 30 °C, the Km values of r-LipA on pNP acetate are 0.037 ± 0.001 and 0.174 ± 0.006 mM, respectively. Higher pH and temperature conditions promoted hydrolytic activity toward the pNP esters with longer chain fatty acids. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents. The results suggest that the r-LipA protein has some new characteristics potentially promising for industrial applications and S. cellulosum is an intriguing resource for lipase screening

Survival in Nuclear Waste, Extreme Resistance, and Potential Applications Gleaned from the Genome Sequence of Kineococcus radiotolerans SRS30216

Kineococcus radiotolerans SRS30216 was isolated from a high-level radioactive environment at the Savannah River Site (SRS) and exhibits γ-radiation resistance approaching that of Deinococcus radiodurans. The genome was sequenced by the U.S. Department of Energy's Joint Genome Institute which suggested the existence of three replicons, a 4.76 Mb linear chromosome, a 0.18 Mb linear plasmid, and a 12.92 Kb circular plasmid. Southern hybridization confirmed that the chromosome is linear. The K. radiotolerans genome sequence was examined to learn about the physiology of the organism with regard to ionizing radiation resistance, the potential for bioremediation of nuclear waste, and the dimorphic life cycle. K. radiotolerans may have a unique genetic toolbox for radiation protection as it lacks many of the genes known to confer radiation resistance in D. radiodurans. Additionally, genes involved in the detoxification of reactive oxygen species and the excision repair pathway are overrepresented. K. radiotolerans appears to lack degradation pathways for pervasive soil and groundwater pollutants. However, it can respire on two organic acids found in SRS high-level nuclear waste, formate and oxalate, which promote the survival of cells during prolonged periods of starvation. The dimorphic life cycle involves the production of motile zoospores. The flagellar biosynthesis genes are located on a motility island, though its regulation could not be fully discerned. These results highlight the remarkable ability of K radiotolerans to withstand environmental extremes and suggest that in situ bioremediation of organic complexants from high level radioactive waste may be feasible

Altered Regulation of Renal Sodium Transporters in Salt-Sensitive Hypertensive Rats Induced by Uninephrectomy

Uninephrectomy (uNx) in young rats causes salt-sensitive hypertension (SSH). Alterations of sodium handling in residual nephrons may play a role in the pathogenesis. Therefore, we evaluated the adaptive alterations of renal sodium transporters according to salt intake in uNx-SSH rats. uNx or sham operations were performed in male Sprague-Dawley rats, and normal-salt diet was fed for 4 weeks. Four experimental groups were used: sham-operated rats raised on a high-salt diet for 2 weeks (CHH) or on a low-salt diet for 1 week after 1 week's high-salt diet (CHL) and uNx rats fed on the same diet (NHH, NHL) as the sham-operated rats were fed. Expression of major renal sodium transporters were determined by semiquantitative immunoblotting. Systolic blood pressure was increased in NHH and NHL groups, compared with CHH and CHL, respectively. Protein abundances of Na+/K+/2Cl- cotransporter (NKCC2) and Na+/Cl- cotransporter (NCC) in the CHH group were lower than the CHL group. Expression of epithelial sodium channel (ENaC)-γ increased in the CHH group. In contrast, expressions of NKCC2 and NCC in the NHH group didn't show any significant alterations, compared to the NHL group. Expressions of ENaC-α and ENaC-β in the NHH group were higher than the CHH group. Adaptive alterations of NKCC2 and NCC to changes of salt intake were different in the uNx group, and changes in ENaC-α and ENaC-β were also different. These altered regulations of sodium transporters may be involved in the pathogenesis of SSH in the uNx rat model

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well‐described rare autosomal dominant condition due to haploinsufficiency of PTCH1 . With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1 . While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non‐BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions. © 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90383/1/34216_ftp.pd

Myxobacteria: Moving, Killing, Feeding, and Surviving Together

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb.2016.00781Myxococcus xanthus, like other myxobacteria, is a social bacterium that moves and feeds cooperatively in predatory groups. On surfaces, rod-shaped vegetative cells move in search of the prey in a coordinated manner, forming dynamic multicellular groups referred to as swarms. Within the swarms, cells interact with one another and use two separate locomotion systems. Adventurous motility, which drives the movement of individual cells, is associated with the secretion of slime that forms trails at the leading edge of the swarms. It has been proposed that cellular traffic along these trails contributes to M. xanthus social behavior via stigmergic regulation. However, most of the cells travel in groups by using social motility, which is cell contact-dependent and requires a large number of individuals. Exopolysaccharides and the retraction of type IV pili at alternate poles of the cells are the engines associated with social motility. When the swarms encounter prey, the population of M. xanthus lyses and takes up nutrients from nearby cells. This cooperative and highly density-dependent feeding behavior has the advantage that the pool of hydrolytic enzymes and other secondary metabolites secreted by the entire group is shared by the community to optimize the use of the degradation products. This multicellular behavior is especially observed in the absence of nutrients. In this condition, M. xanthus swarms have the ability to organize the gliding movements of 1000s of rods, synchronizing rippling waves of oscillating cells, to form macroscopic fruiting bodies, with three subpopulations of cells showing division of labor. A small fraction of cells either develop into resistant myxospores or remain as peripheral rods, while the majority of cells die, probably to provide nutrients to allow aggregation and spore differentiation. Sporulation within multicellular fruiting bodies has the benefit of enabling survival in hostile environments, and increases germination and growth rates when cells encounter favorable conditions. Herein, we review how these social bacteria cooperate and review the main cell–cell signaling systems used for communication to maintain multicellularity.This work has been funded by the Spanish Government (grants CSD2009-00006 and BFU2012-33248, 70% funded by FEDER) and Junta de Andalucía (group BIO318)

Genome Evolution and the Emergence of Fruiting Body Development in Myxococcus xanthus

BACKGROUND: Lateral gene transfer (LGT) is thought to promote speciation in bacteria, though well-defined examples have not been put forward. METHODOLOGY/PRINCIPLE FINDINGS: We examined the evolutionary history of the genes essential for a trait that defines a phylogenetic order, namely fruiting body development of the Myxococcales. Seventy-eight genes that are essential for Myxococcus xanthus development were examined for LGT. About 73% of the genes exhibit a phylogeny similar to that of the 16S rDNA gene and a codon bias consistent with other M. xanthus genes suggesting vertical transmission. About 22% have an altered codon bias and/or phylogeny suggestive of LGT. The remaining 5% are unique. Genes encoding signal production and sensory transduction were more likely to be transmitted vertically with clear examples of duplication and divergence into multigene families. Genes encoding metabolic enzymes were frequently acquired by LGT. Myxobacteria exhibit aerobic respiration unlike most of the delta Proteobacteria. M. xanthus contains a unique electron transport pathway shaped by LGT of genes for succinate dehydrogenase and three cytochrome oxidase complexes. CONCLUSIONS/SIGNIFICANCE: Fruiting body development depends on genes acquired by LGT, particularly those involved in polysaccharide production. We suggest that aerobic growth fostered innovation necessary for development by allowing myxobacteria access to a different gene pool from anaerobic members of the delta Proteobacteria. Habitat destruction and loss of species diversity could restrict the evolution of new bacterial groups by limiting the size of the prospective gene pool

Components of variance in transcriptomics based on electrophoretic separation of cDNA fragments (cDNA-AFLP)

The sources of variance and errors in transcriptomics based on the electrophoretic separation of amplified cDNA fragments were investigated using cDNA-amplified fragment length polymorphism (AFLP). Transcriptome profiles of the plant-pathogenic fungus Verticillium longisporum were generated by a standard cDNA-AFLP protocol followed by electrophoretic separation of amplified DNA fragments in flatbed polyacrylamide gels with fluorescence detection as well as by capillary electrophoresis (DNA sequencer). The total variance was partitioned into contributions of cDNA synthesis, adapter ligation, preamplification, amplification, and electrophoresis. Parameters of computer-aided peak recognition and matching were investigated and strategies improving matching success based on double passage with different signal intensity thresholds were developed. The overall quality of data was similar for cDNA-AFLP and microarray hybridization. Variance of cDNA-AFLP was independent of signal intensity, whereas microarray data showed higher variance for low-intensity signals. Capillary electrophoresis significantly reduced the number of wrongly matched and unmatched signals as compared with flatbed gels. These results are also likely to apply to related electrophoresis-based transcriptome analysis techniques such as mRNA differential display