scholarly journals Identification of Major Sporulation Proteins of Myxococcus xanthus Using a Proteomic Approach

2007 ◽  
Vol 189 (8) ◽  
pp. 3187-3197 ◽  
Author(s):  
John L. Dahl ◽  
Farah K. Tengra ◽  
David Dutton ◽  
Jinyuan Yan ◽  
Tracy M. Andacht ◽  
...  
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Protein S ◽  
Differentially Expressed ◽  
Insertion Mutagenesis ◽  
Wild Type ◽  
Proteomic Approach ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Myxococcus xanthus is a soil-dwelling, gram-negative bacterium that during nutrient deprivation is capable of undergoing morphogenesis from a vegetative rod to a spherical, stress-resistant spore inside a domed-shaped, multicellular fruiting body. To identify proteins required for building stress-resistant M. xanthus spores, we compared the proteome of liquid-grown vegetative cells with the proteome of mature fruiting body spores. Two proteins, protein S and protein S1, were differentially expressed in spores, as has been reported previously. In addition, we identified three previously uncharacterized proteins that are differentially expressed in spores and that exhibit no homology to known proteins. The genes encoding these three novel major spore proteins (mspA, mspB, and mspC) were inactivated by insertion mutagenesis, and the development of the resulting mutant strains was characterized. All three mutants were capable of aggregating, but for two of the strains the resulting fruiting bodies remained flattened mounds of cells. The most pronounced structural defect of spores produced by all three mutants was an altered cortex layer. We found that mspA and mspB mutant spores were more sensitive specifically to heat and sodium dodecyl sulfate than wild-type spores, while mspC mutant spores were more sensitive to all stress treatments examined. Hence, the products of mspA, mspB, and mspC play significant roles in morphogenesis of M. xanthus spores and in the ability of spores to survive environmental stress.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

Murein components rescue developmental sporulation of Myxococcus xanthus

1982 ◽  
Vol 152 (1) ◽  
pp. 462-470 ◽  
Author(s):  
L J Shimkets ◽  
D Kaiser
Keyword(s):  
Fruiting Body ◽  
High Cell Density ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Diaminopimelic Acid ◽  
Nutrient Level ◽  
High Cell ◽  
Wild Type ◽  
Nutrient Rich Medium

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.

scholarly journals Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

1982 ◽  
Vol 152 (1) ◽  
pp. 166-174
Author(s):  
J A Mulder ◽  
G Venema
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Magnesium Ions ◽  
Polyacrylamide Gels ◽  
Wild Type ◽  
Molecular Weights ◽  
Defective Mutant ◽  
Mutant Strains

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

scholarly journals Data-Driven Models Reveal Mutant Cell Behaviors Important for Myxobacterial Aggregation

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Zhaoyang Zhang ◽  
Christopher R. Cotter ◽  
Zhe Lyu ◽  
Lawrence J. Shimkets ◽  
Oleg A. Igoshin
Keyword(s):  
Cell Tracking ◽  
Mutant Cell ◽  
Myxococcus Xanthus ◽  
Data Driven ◽  
Self Organization ◽  
Cell Behavior ◽  
Significant Heterogeneity ◽  
Wild Type ◽  
Content Type ◽  
Mutant Strains

ABSTRACT Single mutations frequently alter several aspects of cell behavior but rarely reveal whether a particular statistically significant change is biologically significant. To determine which behavioral changes are most important for multicellular self-organization, we devised a new methodology using Myxococcus xanthus as a model system. During development, myxobacteria coordinate their movement to aggregate into spore-filled fruiting bodies. We investigate how aggregation is restored in two mutants, csgA and pilC, that cannot aggregate unless mixed with wild-type (WT) cells. To this end, we use cell tracking to follow the movement of fluorescently labeled cells in combination with data-driven agent-based modeling. The results indicate that just like WT cells, both mutants bias their movement toward aggregates and reduce motility inside aggregates. However, several aspects of mutant behavior remain uncorrected by WT, demonstrating that perfect recreation of WT behavior is unnecessary. In fact, synergies between errant behaviors can make aggregation robust. IMPORTANCE Self-organization into spatial patterns is evident in many multicellular phenomena. Even for the best-studied systems, our ability to dissect the mechanisms driving coordinated cell movement is limited. While genetic approaches can identify mutations perturbing multicellular patterns, the diverse nature of the signaling cues coupled to significant heterogeneity of individual cell behavior impedes our ability to mechanistically connect genes with phenotype. Small differences in the behaviors of mutant strains could be irrelevant or could sometimes lead to large differences in the emergent patterns. Here, we investigate rescue of multicellular aggregation in two mutant strains of Myxococcus xanthus mixed with wild-type cells. The results demonstrate how careful quantification of cell behavior coupled to data-driven modeling can identify specific motility features responsible for cell aggregation and thereby reveal important synergies and compensatory mechanisms. Notably, mutant cells do not need to precisely recreate wild-type behaviors to achieve complete aggregation.

scholarly journals Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

2012 ◽  
Vol 11 (5) ◽  
pp. 694-702 ◽  
Author(s):  
Ahmed Hamam ◽  
Roger R. Lew
Keyword(s):  
Atpase Activity ◽  
Calcium Transport ◽  
Tip Growth ◽  
Electrical Characterization ◽  
Mechanosensitive Channel ◽  
Wild Type ◽  
Current Voltage ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Hyphal Tip

ABSTRACT We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog ( MscS ), a Ca 2+ /H + exchange protein ( cax ), and Ca 2+ -ATPases ( nca-1 , nca-2 , nca-3 )—as well as those of double mutants (the nca-2 cax , nca-2 nca-3 , and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2 -containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H + -ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca 2+ levels, indicative of lesions in Ca 2+ homeostasis. However, the net Ca 2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca 2+ -selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca 2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca 2+ ] was elevated. Thus, although Ca 2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H + -ATPase activity.

scholarly journals Proteins Associated with the Myxococcus xanthus Extracellular Matrix

2007 ◽  
Vol 189 (21) ◽  
pp. 7634-7642 ◽  
Author(s):  
Patrick D. Curtis ◽  
James Atwood ◽  
Ron Orlando ◽  
Lawrence J. Shimkets
Keyword(s):  
Extracellular Matrix ◽  
Biofilm Formation ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Fruiting Body Formation ◽  
Ecm Proteins ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass ◽  
In The Wild

ABSTRACT Fruiting body formation of Myxococcus xanthus, like biofilm formation of many other organisms, involves the production of an extracellular matrix (ECM). While the polysaccharide component has been studied, the protein component has been largely unexplored. Proteins associated with the ECM were solubilized from purified ECM by boiling with sodium dodecyl sulfate and were identified by liquid chromatography-tandem mass spectrometry of tryptic fragments. The ECM is enriched in proteins of novel function; putative functions were assigned for only 5 of the 21 proteins. Thirteen putative ECM proteins had lipoprotein secretion signals. The genes for many ECM proteins were disrupted in the wild-type (WT), fibA, and pilA backgrounds. Disruption of the MXAN4860 gene had no effect in the WT or fibA background but in the pilA background resulted in a 24-h delay in aggregation and sporulation compared to its parent. The results of this study show that the M. xanthus ECM proteome is diverse and novel.

scholarly journals Two Lipid Signals Guide Fruiting Body Development of Myxococcus xanthus

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Swapna Bhat ◽  
Tilman Ahrendt ◽  
Christina Dauth ◽  
Helge B. Bode ◽  
Lawrence J. Shimkets
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
Chain Fatty Acid ◽  
Fatty Alcohols ◽  
Lipid Bodies ◽  
Wild Type ◽  
Bioactive Lipids ◽  
Content Type

ABSTRACTMyxococcus xanthusproduces several extracellular signals that guide fruiting body morphogenesis and spore differentiation. Mutants defective in producing a signal may be rescued by codevelopment with wild-type cells or cell fractions containing the signal. In this paper, we identify two molecules that rescue development of the E signal-deficient mutant LS1191 at physiological concentrations,iso15:0 branched-chain fatty acid (FA) and 1-iso15:0-alkyl-2,3-di-iso15:0-acyl glycerol (TG1), a development-specific monoalkyl-diacylglycerol. The physiological concentrations of the bioactive lipids were determined by mass spectrometry from developing wild-type cells using chemically synthesized standards. Synthetic TG1 restored fruiting body morphogenesis and sporulation and activated the expression of the developmentally regulated gene with locus tagMXAN_2146at physiological concentrations, unlike its nearly identical tri-iso15:0 triacylglycerol (TAG) counterpart, which has an ester linkage instead of an ether linkage.iso15:0 FA restored development at physiological concentrations, unlike palmitic acid, a straight-chain fatty acid. The addition of either lipid stimulates cell shortening, with an 87% decline in membrane surface area, concomitantly with the production of lipid bodies at each cell pole and in the center of the cell. We suggest that cells produce triacylglycerol from membrane phospholipids. Bioactive lipids may be released byprogrammedcelldeath (PCD), which claims up to 80% of developing cells, since cells undergoing PCD produce lipid bodies before lysing.IMPORTANCELike mammalian adipose tissue, many of theM. xanthuslipid body lipids are triacylglycerols (TAGs), containing ester-linked fatty acids. In both systems, ester-linked fatty acids are retrieved from TAGs with lipases and consumed by the fatty acid degradation cycle. Both mammals andM. xanthusalso produce lipids containing ether-linked fatty alcohols with alkyl or vinyl linkages, such as plasmalogens. Alkyl and vinyl linkages are not hydrolyzed by lipases, and no clear role has emerged for lipids bearing them. For example, plasmalogen deficiency in mice has detrimental consequences to spermatocyte development, myelination, axonal survival, eye development, and long-term survival, though the precise reasons remain elusive. Lipids containing alkyl- and vinyl-linked fatty alcohols are development-specific products inM. xanthus. Here, we show that one of them rescues the development of E signal-producing mutants at physiological concentrations.

EnteropathogenicEscherichia coliinhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

2006 ◽  
Vol 290 (1) ◽  
pp. G30-G35 ◽  
Author(s):  
Alip Borthakur ◽  
Ravinder K. Gill ◽  
Kim Hodges ◽  
Krishnamurthy Ramaswamy ◽  
Gail Hecht ◽  
...  
Keyword(s):  
Surface Expression ◽  
Monocarboxylate Transporter ◽  
Wild Type ◽  
Infantile Diarrhea ◽  
Monocarboxylate Transporter 1 ◽  
E Coli ◽  
Food Borne ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Fatty Acid Absorption

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+and Cl−/HCO3−exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.

On the Evolution of an Oligocephalic Enzyme. Glutamine- Chorismate-Amidotransferase-Free Anthranilate Phosphoribosyltransferases from Mutant Strains of Salmonella typhimurium

1978 ◽  
Vol 33 (3-4) ◽  
pp. 235-244 ◽  
Author(s):  
Manfred Grieshaber
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulfate ◽  
Salmonella Typhimurium ◽  
Sodium Dodecyl ◽  
Single Component ◽  
Wild Type ◽  
Molecular Weights ◽  
Mutant Strains ◽  
Dodecyl Sulfate

(1) A procedure has been described for the purification of two glutamine-chorismate-amido- transferase-free anthranilate phosphoribosyltransferases from mutant strains TAX6trpR782 and trpAB1653trpR782 of Salmonella typhimurium.(2) The native enzymes tend to aggregate forming polymers of molecular weights 333,000 in the case of TAXtrpR782 and 220,000 and larger than 1X106 in the case of trpABI653trpR782. In the presence of sodium dodecyl sulfate the polymer of trpAB1653trpR782 dissociates into a single component with molecular weight of 72,000.(3) In contrast to anthranilate phosphoribosyltransferase of the wild type component II, the glutamine-chorismate-amidotransferase-free proteins do not complex with component I. They do however show catalytical similarities with the wild type with respect to anthranilate phosphoribosyltransferase activity.

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