scholarly journals A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus

1998 ◽  
Vol 180 (20) ◽  
pp. 5357-5368 ◽  
Author(s):  
Robby M. Weimer ◽  
Chad Creighton ◽  
Angela Stassinopoulos ◽  
Philip Youderian ◽  
Patricia L. Hartzell
Keyword(s):  
Myxococcus Xanthus ◽  
Extracellular Polysaccharide ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Multicellular Development ◽  
Hsp70 Family ◽  
Alginate Production ◽  
Social Motility ◽  
Extracellular Fibrils ◽  
Chaperone Hsp70

ABSTRACT Three independent Tn5-lac insertions in the S1 locus ofMyxococcus xanthus inactivate the sglK gene, which is nonessential for growth but required for social motility and multicellular development. The sequence of sglK reveals that it encodes a homologue of the chaperone HSP70 (DnaK). ThesglK gene is cotranscribed with the upstreamgrpS gene, which encodes a GrpE homologue. UnlikesglK, grpS is not required for social motility or development. Wild-type M. xanthus is encased in extracellular polysaccharide filaments associated with the multimeric fibrillin protein. Mutations in sglK inhibit cell cohesion, the binding of Congo red, and the synthesis or secretion of fibrillin, indicating that sglK mutants do not make fibrils. ThefibR gene, located immediately upstream of thegrpS-sglK operon, encodes a product which is predicted to have a sequence similar to those of the repressors of alginate biosynthesis in Pseudomonas aeruginosa andPseudomonas putida. Inactivation of fibR leads to the overproduction of fibrillin, suggesting that M. xanthus fibril production and Pseudomonas alginate production are regulated in analogous ways. M. xanthus andPseudomonas exopolysaccharides may play similar roles in a mechanism of social motility conserved in these gram-negative bacteria.

scholarly journals Novel Developmental Genes, fruCD, of Myxococcus xanthus: Involvement of a Cell Division Protein in Multicellular Development

2003 ◽  
Vol 185 (11) ◽  
pp. 3317-3324 ◽  
Author(s):  
Takuya Akiyama ◽  
Sumiko Inouye ◽  
Teruya Komano
Keyword(s):  
Vegetative Growth ◽  
Transcription Initiation ◽  
Growth And Development ◽  
Sequence Similarity ◽  
Myxococcus Xanthus ◽  
Spore Formation ◽  
Amino Acid Sequence Similarity ◽  
Wild Type ◽  
Developmental Genes ◽  
Multicellular Development

ABSTRACT Myxococcus xanthus is a gram-negative soil bacterium that undergoes multicellular development upon nutrient starvation. In the present study, two novel developmental genes, fruC and fruD, of M. xanthus were identified and characterized. The FruD protein has significant amino acid sequence similarity to the DivIVA proteins of many bacteria including Bacillus subtilis. Vegetative cells of the fruD mutant exhibited a filamentous phenotype. The fruC and fruD mutants displayed similar delayed-development phenotypes. The formation of tightly aggregated mounds by fruC and fruD mutants was slower than that by the wild-type strain. Spore formation by the fruC and fruD mutants initiated after 30 h poststarvation, whereas wild-type M. xanthus initiated spore formation after 18 h. The fruCD genes were constitutively expressed as an operon during vegetative growth and development. S1 mapping revealed that transcription initiation sites of the fruCD operon were located 114 (P1) and 55 bp (P2) upstream of the fruC initiation codon. Only the P1 promoter was active during vegetative growth, while both the P1 and P2 promoters were active during development. The FruD protein was produced as a cytoplasmic protein and formed an oligomer during vegetative growth and development.

scholarly journals Contact Stimulation of Tgl and Type IV Pili inMyxococcus xanthus

1998 ◽  
Vol 180 (3) ◽  
pp. 759-761 ◽  
Author(s):  
Daniel Wall ◽  
Samuel S. Wu ◽  
Dale Kaiser
Keyword(s):  
Myxococcus Xanthus ◽  
Type Iv Pili ◽  
Wild Type ◽  
Rapid Assay ◽  
Type Iv ◽  
Social Motility ◽  
Stimulation Of

ABSTRACT Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl + cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.

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.

scholarly journals Role of Autocleavage in the Function of a Type III Secretion Specificity Switch Protein in Salmonella enterica Serovar Typhimurium

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Julia V. Monjarás Feria ◽  
Matthew D. Lefebre ◽  
York-Dieter Stierhof ◽  
Jorge E. Galán ◽  
Samuel Wagner
Keyword(s):  
Host Cell ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Switching Function ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Type Iii ◽  
Autocatalytic Cleavage ◽  
Serovar Typhimurium

ABSTRACTType III secretion systems (T3SSs) are multiprotein machines employed by many Gram-negative bacteria to inject bacterial effector proteins into eukaryotic host cells to promote bacterial survival and colonization. The core unit of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins through the bacterial envelope. A distinct feature of the T3SS is that protein export occurs in a strictly hierarchical manner in which proteins destined to form the needle complex filament and associated structures are secreted first, followed by the secretion of effectors and the proteins that will facilitate their translocation through the target host cell membrane. The secretion hierarchy is established by complex mechanisms that involve several T3SS-associated components, including the “switch protein,” a highly conserved, inner membrane protease that undergoes autocatalytic cleavage. It has been proposed that the autocleavage of the switch protein is the trigger for substrate switching. We show here that autocleavage of theSalmonella entericaserovar Typhimurium switch protein SpaS is an unregulated process that occurs after its folding and before its incorporation into the needle complex. Needle complexes assembled with a precleaved form of SpaS function in a manner indistinguishable from that of the wild-type form. Furthermore, an engineered mutant of SpaS that is processed by an external protease also displays wild-type function. These results demonstrate that the cleavage eventper sedoes not provide a signal for substrate switching but support the hypothesis that cleavage allows the proper conformation of SpaS to render it competent for its switching function.IMPORTANCEBacterial interaction with eukaryotic hosts often involves complex molecular machines for targeted delivery of bacterial effector proteins. One such machine, the type III secretion system of some Gram-negative bacteria, serves to inject a multitude of structurally diverse bacterial proteins into the host cell. Critical to the function of these systems is their ability to secrete proteins in a strict hierarchical order, but it is unclear how the mechanism of switching works. Central to the switching mechanism is a highly conserved inner membrane protease that undergoes autocatalytic cleavage. Although it has been suggested previously that the autocleavage event is the trigger for substrate switching, we show here that this is not the case. Rather, our results show that cleavage allows the proper conformation of the protein to render it competent for its switching function. These findings may help develop inhibitors of type III secretion machines that offer novel therapeutic avenues to treat various infectious diseases.

scholarly journals The role of membrane destabilisation and protein dynamics in BAM catalysed OMP folding

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Paul White ◽  
Samuel F. Haysom ◽  
Matthew G. Iadanza ◽  
Anna J. Higgins ◽  
Jonathan M. Machin ◽  
...  
Keyword(s):  
Outer Membrane Proteins ◽  
Antibody Fragment ◽  
Membrane Disruption ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Lipid Dynamics ◽  
Open Structures

AbstractThe folding of β-barrel outer membrane proteins (OMPs) in Gram-negative bacteria is catalysed by the β-barrel assembly machinery (BAM). How lateral opening in the β-barrel of the major subunit BamA assists in OMP folding, and the contribution of membrane disruption to BAM catalysis remain unresolved. Here, we use an anti-BamA monoclonal antibody fragment (Fab1) and two disulphide-crosslinked BAM variants (lid-locked (LL), and POTRA-5-locked (P5L)) to dissect these roles. Despite being lethal in vivo, we show that all complexes catalyse folding in vitro, albeit less efficiently than wild-type BAM. CryoEM reveals that while Fab1 and BAM-P5L trap an open-barrel state, BAM-LL contains a mixture of closed and contorted, partially-open structures. Finally, all three complexes globally destabilise the lipid bilayer, while BamA does not, revealing that the BAM lipoproteins are required for this function. Together the results provide insights into the role of BAM structure and lipid dynamics in OMP folding.

scholarly journals The kfrA gene is the first in a tricistronic operon required for survival of IncP-1 plasmid R751

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1621-1637 ◽  
Author(s):  
Malgorzata Adamczyk ◽  
Patrycja Dolowy ◽  
Michal Jonczyk ◽  
Christopher M. Thomas ◽  
Grazyna Jagura-Burdzy
Keyword(s):  
Coiled Coil ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
C Terminus ◽  
Low Copy Number ◽  
And Control ◽  
Broad Host Range Plasmids

The kfrA gene of the IncP-1 broad-host-range plasmids is the best-studied member of a growing gene family that shows strong linkage to the minimal replicon of many low-copy-number plasmids. KfrA is a DNA binding protein with a long, alpha-helical, coiled-coil tail. Studying IncP-1β plasmid R751, evidence is presented that kfrA and its downstream genes upf54.8 and upf54.4 were organized in a tricistronic operon (renamed here kfrA kfrB kfrC), expressed from autoregulated kfrAp, that was also repressed by KorA and KorB. KfrA, KfrB and KfrC interacted and may have formed a multi-protein complex. Inactivation of either kfrA or kfrB in R751 resulted in long-term accumulation of plasmid-negative bacteria, whereas wild-type R751 itself persisted without selection. Immunofluorescence studies showed that KfrAR751 formed plasmid-associated foci, and deletion of the C terminus of KfrA caused plasmid R751ΔC 2 kfrA foci to disperse and mislocalize. Thus, the KfrABC complex may be an important component in the organization and control of the plasmid clusters that seem to form the segregating unit in bacterial cells. The studied operon is therefore part of the set of functions needed for R751 to function as an efficient vehicle for maintenance and spread of genes in Gram-negative bacteria.

scholarly journals Mining of Gram-Negative Surface-Active Enzybiotic Candidates by Sequence-Based Calculation of Physicochemical Properties

2021 ◽  
Vol 12 ◽  
Author(s):  
Roberto Vázquez ◽  
Sofía Blanco-Gañán ◽  
Susana Ruiz ◽  
Pedro García
Keyword(s):  
Physicochemical Properties ◽  
Nearest Neighbors ◽  
Regulatory Agencies ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Bacterial Surface ◽  
Negative Surface ◽  
Surface Active ◽  
Phage Lysins

Phage (endo)lysins are nowadays one of the most promising ways out of the current antibiotic resistance crisis. Either as sole therapeutics or as a complement to common antibiotic chemotherapy, lysins are already entering late clinical phases to get regulatory agencies’ authorization. Even the old paradigm of the inability of lysins to attack Gram-negative bacteria from without has already been overcome in a variety of ways: either by engineering approaches or investigating the natural mechanisms by which some wild-type lysins are able to interact with the bacterial surface. Such inherent ability of some lysins has been linked to antimicrobial peptide (AMP)-like regions, which are, on their own, a significant source for novel antimicrobials. Currently, though, many of the efforts for searching novel lysin-based antimicrobial candidates rely on experimental screenings. In this work, we have bioinformatically analyzed the C-terminal end of a collection of lysins from phages infecting the Gram-negative genus Pseudomonas. Through the computation of physicochemical properties, the probability of such regions to be an AMP was estimated by means of a predictive k-nearest neighbors (kNN) model. This way, a subset of putatively membrane-interacting lysins was obtained from the original database. Two of such candidates (named Pae87 and Ppl65) were prospectively tested in terms of muralytic, bacteriolytic, and bactericidal activity. Both of them were found to possess an activity against Pseudomonas aeruginosa and other Gram-negative bacterial pathogens, implying that the prediction of AMP-like regions could be a useful approach toward the mining of phage lysins to design and develop antimicrobials or antimicrobial parts for further engineering.

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.

Mutants lacking myosin II cannot resist forces generated during multicellular morphogenesis

1995 ◽  
Vol 108 (3) ◽  
pp. 1105-1115 ◽  
Author(s):  
E. Shelden ◽  
D.A. Knecht
Keyword(s):  
Cell Morphology ◽  
Mutant Cell ◽  
Myosin Ii ◽  
Cell Phenotype ◽  
Computer Assisted ◽  
Wild Type ◽  
Isolated Cells ◽  
Multicellular Development ◽  
Periodic Movement ◽  
Mutant Cells

We have used fluorescent labeling, confocal microscopy and computer-assisted motion analysis to observe and quantify individual wild-type and myosin II mutant cell behavior during early multicellular development in Dictyostelium discoideum. When cultured with an excess of unlabeled wild-type cells, labeled control cells are randomly distributed within aggregation streams, while myosin II mutant cells are found primarily at the lateral edges of streams. Wild-type cells move at average rates of 8.5 +/- 4.9 microns/min within aggregation streams and can exhibit regular periodic movement at 3.5 minute intervals; half as long as the 7 minute period reported previously for isolated cells. Myosin II mutants under the same conditions move at 5.0 +/- 4.8 microns/min, twice as fast as reported previously for isolated myosin II mutant cells, and fail to display regular periodic movement. When removed from aggregation streams myosin II mutant cells move at only 2.5 +/- 2.0 microns/min, while wild-type cells under these conditions move at 5.9 +/- 4.5 microns/min. Analysis of cell morphology further reveals that myosin II mutant cells are grossly and dynamically deformed within wild-type aggregation streams but not when removed from streams and examined in isolation. These data reveal that the loss of myosin II has dramatic consequences for cells undergoing multicellular development. The segregation of mutant cells to aggregation stream edges demonstrates that myosin II mutants are unable to penetrate a multicellular mass of wild-type cells, while the observed distortion of myosin II mutant cells suggests that the cortex of such cells is too flacid to resist forces generated during movement. The increased rate of mutant cell movement and distortion of mutant cell morphology seen within wild-type aggregation streams further argues both that movement of wild-type cells within a multicellular mass can generate traction forces on neighboring cells and that mutant cell morphology and behavior can be altered by these forces. In addition, the distortion of myosin II mutant cells within wild-type aggregation streams indicates that myosin is not required for the formation of cell-cell contacts. Finally, the consequences of the loss of myosin II for cells during multicellular development are much more severe than has been previously revealed for isolated cells. The techniques used here to analyze the behavior of individual cells within multicellular aggregates provide a more sensitive assay of mutant cell phenotype than has been previously available and will be generally applicable to the study of motility and cytoskeletal mutants in Dictyostelium.

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