scholarly journals Functional and Mutational Analysis of P19, a DNA Transfer Protein with Muramidase Activity

2001 ◽  
Vol 183 (10) ◽  
pp. 3176-3183 ◽  
Author(s):  
Michaela Bayer ◽  
Robert Iberer ◽  
Karin Bischof ◽  
Edith Rassi ◽  
Edith Stabentheiner ◽  
...  
Keyword(s):  
Mutational Analysis ◽  
Cell Envelope ◽  
Large Family ◽  
Conjugative Plasmid ◽  
Secretion Systems ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Plasmid R1 ◽  
Muramidase Activity

ABSTRACT Protein P19 encoded by the conjugative resistance plasmid R1 has been identified as being one member of a large family of muramidases encoded by bacteriophages and by type III and type IV secretion systems. We carried out a mutational analysis to investigate the function of protein P19 and used in vivo complementation assays to test those of several P19 mutants. The results indicated that conserved residues present in the presumed catalytic center of P19 are absolutely essential for its function in conjugation of plasmid R1 and infection by the RNA phage R17. Overexpression of protein P19 in an early growth phase resulted in a massive lysis of Escherichia coli cells in liquid culture, as indicated by a rapid and distinct decrease in cell culture densities after induction. Change of the proposed catalytic glutamate at position 44 to glutamine completely abolished this effect. P19-induced cell lysis was directly shown by transmission and scanning electron microscopy. Typically, P19-overexpressing cells showed bulges protruding from the cell surfaces. Our interpretation is that these protrusions arose from a localized and spatially confined disruption of the bacterial cell wall. To our knowledge such an effect has not previously been documented for any member of the lytic transglycosylase family. From the data presented here, we conclude that protein P19 possesses the proposed localized peptidoglycan-hydrolyzing activity. This activity would be a prerequisite for efficient penetration of the cell envelope by the DNA translocation complex encoded by the conjugative plasmid.

scholarly journals Functional and Mutational Analysis of Conjugative Transfer Region 2 (Tra2) from the IncHI1 Plasmid R27

2003 ◽  
Vol 185 (2) ◽  
pp. 581-591 ◽  
Author(s):  
Trevor D. Lawley ◽  
Matthew W. Gilmour ◽  
James E. Gunton ◽  
Dobryan M. Tracz ◽  
Diane E. Taylor
Keyword(s):  
Insertional Mutagenesis ◽  
Mutational Analysis ◽  
Conjugative Plasmid ◽  
Conjugative Transfer ◽  
Transfer System ◽  
Mating Pair ◽  
Secretion Systems ◽  
Transfer Frequency ◽  
Type Iv ◽  
Transfer Region

ABSTRACT The transfer 2 region (Tra2) of the conjugative plasmid drR27 (derepressed R27) was analyzed by PSI-BLAST, insertional mutagenesis, genetic complementation, and an H-pilus assay. Tra2 contains 11 mating-pair formation (Mpf) genes that are essential for conjugative transfer, 9 of which are essential for H-pilus production (trhA, -L, -E, -K, -B, -V, -C, -P, and -W). TrhK has similarity to secretin proteins, suggesting a mechanism by which DNA could traverse the outer membrane of donors. The remaining two Mpf genes, trhU and trhN, play an auxiliary role in H-pilus synthesis and are proposed to be involved in DNA transfer and mating-pair stabilization, respectively. Conjugative transfer abilities were restored for each mutant when complemented with the corresponding transfer gene. In addition to the essential Mpf genes, three genes, trhO, trhZ, and htdA, modulate R27 transfer frequency. Disruption of trhO and trhZ severely reduced the transfer frequencies of drR27, whereas disruption of htdA greatly increased the transfer frequency of wild-type R27 to drR27 levels. A comparison of the essential transfer genes encoded by the Tra2 and Tra1 (T. D. Lawley, M. W. Gilmour, J. E. Gunton, L. J. Standeven, and D. E. Taylor, J. Bacteriol. 184:2173-2183, 2002) of R27 to other transfer systems illustrates that the R27 conjugative transfer system is a chimera composed of IncF-like and IncP-like transfer systems. Furthermore, the Mpf/type IV secretion systems encoded by IncH and IncF transfer systems are distinct from that of the IncP transfer system. The phenotypic and ecological significance of these observations is discussed.

scholarly journals Peptidoglycan degradation by specialized lytic transglycosylases associated with type III and type IV secretion systems

Microbiology ◽  
2005 ◽  
Vol 151 (11) ◽  
pp. 3455-3467 ◽  
Author(s):  
Doris Zahrl ◽  
Maria Wagner ◽  
Karin Bischof ◽  
Michaela Bayer ◽  
Barbara Zavecz ◽  
...  
Keyword(s):  
Secretion System ◽  
Type Iv Secretion ◽  
Conjugative Plasmid ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Lytic Transglycosylases ◽  
Type Iii ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Type Iv Secretion Systems

Specialized lytic transglycosylases are muramidases capable of locally degrading the peptidoglycan meshwork of Gram-negative bacteria. Specialized lytic transglycosylase genes are present in clusters encoding diverse macromolecular transport systems. This paper reports the analysis of selected members of the specialized lytic transglycosylase family from type III and type IV secretion systems. These proteins were analysed in vivo by assaying their ability to complement the DNA transfer defect of the conjugative F-like plasmid R1-16 lacking a functional P19 protein, the specialized lytic transglycosylase of this type IV secretion system. Heterologous complementation was accomplished using IpgF from the plasmid-encoded type III secretion system of Shigella sonnei and TrbN from the type IV secretion system of the conjugative plasmid RP4. In contrast, neither VirB1 proteins (Agrobacterium tumefaciens, Brucella suis) nor IagB (Salmonella enterica) could functionally replace P19. In vitro, IpgF, IagB, both VirB1 proteins, HP0523 (Helicobacter pylori) and P19 displayed peptidoglycanase activity in zymogram analyses. Using an established test system and a newly developed assay it was shown that IpgF degraded peptidoglycan in solution. IpgF was active only after removal of the chaperonin GroEL, which co-purified with IpgF and inhibited its enzymic activity. A mutant IpgF protein in which the predicted catalytic amino acid, Glu42, was replaced by Gln, was completely inactive. IpgF-catalysed peptidoglycan degradation was optimal at pH 6 and was inhibited by the lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A.

scholarly journals Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

2009 ◽  
Vol 191 (22) ◽  
pp. 6877-6887 ◽  
Author(s):  
Sanja Mihajlovic ◽  
Silvia Lang ◽  
Marta V. Sut ◽  
Heimo Strohmaier ◽  
Christian J. Gruber ◽  
...  
Keyword(s):  
Plasmid Dna ◽  
Molecular Mechanisms ◽  
Substrate Uptake ◽  
Secretion Systems ◽  
Type Iv ◽  
Plasmid R1 ◽  
Regulatory Effects ◽  
Dna Processing

ABSTRACT Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔLk = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.

scholarly journals Mutational Analysis of Bacillus megaterium QM B1551 Cortex-Lytic Enzymes

2010 ◽  
Vol 192 (20) ◽  
pp. 5378-5389 ◽  
Author(s):  
Graham Christie ◽  
Fatma Isık Üstok ◽  
Qiaozhi Lu ◽  
Len C. Packman ◽  
Christopher R. Lowe
Keyword(s):  
Bacillus Megaterium ◽  
Mutational Analysis ◽  
Surrounding Medium ◽  
Molecular Genetic ◽  
Lytic Enzymes ◽  
Analysis Techniques ◽  
Terminal Domain ◽  
Lytic Transglycosylase ◽  
Mutant Strains

ABSTRACT Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.

scholarly journals AtlA Functions as a Peptidoglycan Lytic Transglycosylase in the Neisseria gonorrhoeae Type IV Secretion System

2007 ◽  
Vol 189 (15) ◽  
pp. 5421-5428 ◽  
Author(s):  
Petra L. Kohler ◽  
Holly L. Hamilton ◽  
Karen Cloud-Hansen ◽  
Joseph P. Dillard
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Lytic Transglycosylases ◽  
Type Iv ◽  
Lytic Transglycosylase ◽  
Enzymatic Function ◽  
Type Iv Secretion Systems

ABSTRACT Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

scholarly journals Enterococcal PrgA extends far outside the cell and provides surface exclusion to protect against unwanted conjugation

2020 ◽  
Author(s):  
Andreas Schmitt ◽  
Helmut Hirt ◽  
Michael A. Järvå ◽  
Wei-Sheng Sun ◽  
Josy ter Beek ◽  
...  
Keyword(s):  
Surface Protein ◽  
Cellular Adhesion ◽  
Conjugative Plasmid ◽  
Secretion Systems ◽  
Protease Domain ◽  
Fitness Advantage ◽  
Cellular Aggregation ◽  
Order Of Magnitude ◽  
Surface Exclusion

AbstractHorizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a 2nd copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

scholarly journals In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center at the Channel Entrance

2021 ◽  
Author(s):  
Pratick Khara ◽  
Peter J. Christie ◽  
Bo Hu
Keyword(s):  
Cell Envelope ◽  
Secretion System ◽  
Type Iv Secretion ◽  
Type Iv Secretion System ◽  
Membrane Complex ◽  
Type Iv ◽  
Inner Membrane Complex ◽  
Cytoplasmic Complex

Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as ‘minimized’ or ‘expanded’ based on whether assembly requires only a core set of signature subunits or additional system-specific components. The prototypical ‘minimized’ systems mediating Agrobacterium tumefaciens T-DNA transfer and conjugative transfer of plasmids pKM101 and R388 are built from 12 subunits generically named VirB1-VirB11 and VirD4. In this study, we visualized the pKM101-encoded T4SS in the native context of the bacterial cell envelope by in situ cryoelectron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCCexhibits 14-fold symmetry and resembles that of the T4SSR388, a large substructure of which was previously purified and analyzed by negative-stain electron microscopy (nsEM). The IMC of the in situ T4SSpKM101 machine is highly symmetrical and exhibits 6-fold symmetry, dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMCclosely resembles equivalent regions of three ‘expanded’ T4SSs previously visualized by in situ CryoET, but strikingly differs from the IMC of the purified T4SSR388 whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers.  Together, our findings support a unified architectural model for all T4SSs assembled in vivo regardless of their classification as ‘minimized’ or ‘expanded’: the signature VirB4-like ATPases invariably are arranged as central hexamers of dimers at the entrances to the T4SS channels.

scholarly journals Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters

2009 ◽  
Vol 192 (1) ◽  
pp. 217-224 ◽  
Author(s):  
Rodrigo Sieira ◽  
Gastón M. Arocena ◽  
Lucas Bukata ◽  
Diego J. Comerci ◽  
Rodolfo A. Ugalde
Keyword(s):  
Brucella Abortus ◽  
Cell Envelope ◽  
Host Cells ◽  
Adaptive Responses ◽  
Secretion Systems ◽  
Mobility Shift ◽  
Type Iv ◽  
Dnase I Footprinting ◽  
Direct Binding ◽  
Utilization Pathway

ABSTRACT Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (P virB ). Using different procedures, we isolated a 27-kDa protein that binds specifically to P virB . This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses.

scholarly journals Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1

FEBS Letters ◽  
2000 ◽  
Vol 466 (2-3) ◽  
pp. 389-393 ◽  
Author(s):  
Michaela Bayer ◽  
Karin Bischof ◽  
Rainer Noiges ◽  
Günther Koraimann
Keyword(s):  
Subcellular Localization ◽  
Conjugative Plasmid ◽  
Lytic Transglycosylase ◽  
Plasmid R1

scholarly journals Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes

2021 ◽  
Vol 12 ◽  
Author(s):  
Simon Czolkoss ◽  
Xenia Safronov ◽  
Sascha Rexroth ◽  
Lisa R. Knoke ◽  
Meriyem Aktas ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Cellular Localization ◽  
Cell Envelope ◽  
Label Free ◽  
Secretion Systems ◽  
Type Iv ◽  
Type Vi Secretion ◽  
Marker Proteins ◽  
Associated Proteins ◽  
Detergent Resistant Membranes

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.

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