In vivo architecture of the polar organizing protein Z (PopZ) meshwork in the Alphaproteobacteria Magnetospirillum gryphiswaldense and Caulobacter crescentus

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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A comprehensive assessment of the biocompatibility of Magnetospirillum gryphiswaldense MSR-1 bacterial magnetosomes in vitro and in vivo

Toxicology ◽

10.1016/j.tox.2021.152949 ◽

2021 ◽

Vol 462 ◽

pp. 152949

Author(s):

Xiaohui Nan ◽

Yan Teng ◽

Jiesheng Tian ◽

Zhiyuan Hu ◽

Qiaojun Fang

Keyword(s):

Comprehensive Assessment ◽

Magnetospirillum Gryphiswaldense

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Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

Molecular Microbiology ◽

10.1046/j.1365-2958.2001.02285.x ◽

2001 ◽

Vol 39 (4) ◽

pp. 924-934 ◽

Cited By ~ 22

Author(s):

B. Lin ◽

J. M. Skidmore ◽

A. Bhatt ◽

S. M. Pfeffer ◽

L. Pawloski ◽

...

Keyword(s):

Amino Acids ◽

Caulobacter Crescentus ◽

Alanine Scan

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Proteolytic Adaptor for Transfer-Messenger RNA-Tagged Proteins from α-Proteobacteria

Journal of Bacteriology ◽

10.1128/jb.01387-06 ◽

2006 ◽

Vol 189 (1) ◽

pp. 272-275 ◽

Cited By ~ 21

Author(s):

Faith H. Lessner ◽

Bryan J. Venters ◽

Kenneth C. Keiler

Keyword(s):

Escherichia Coli ◽

Messenger Rna ◽

Caulobacter Crescentus ◽

Sequence Similarity ◽

Limited Sequence

ABSTRACT We have identified an analog of SspB, the proteolytic adaptor for transfer-messenger RNA (tmRNA)-tagged proteins, in Caulobacter crescentus. C. crescentus SspB shares limited sequence similarity with Escherichia coli SspB but binds the tmRNA tag in vitro and is required for optimal proteolysis of tagged proteins in vivo.

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The Core Dimerization Domains of Histidine Kinases Contain Recognition Specificity for the Cognate Response Regulator

Journal of Bacteriology ◽

10.1128/jb.185.15.4424-4431.2003 ◽

2003 ◽

Vol 185 (15) ◽

pp. 4424-4431 ◽

Cited By ~ 44

Author(s):

Noriko Ohta ◽

Austin Newton

Keyword(s):

Signal Transduction ◽

Caulobacter Crescentus ◽

Response Regulator ◽

Signal Transduction Pathways ◽

Histidine Kinases ◽

Helix Bundle ◽

Recognition Specificity ◽

Four Helix Bundle ◽

Two Hybrid

ABSTRACT Histidine kinases DivJ and PleC initiate signal transduction pathways that regulate an early cell division cycle step and the gain of motility later in the Caulobacter crescentus cell cycle, respectively. The essential single-domain response regulator DivK functions downstream of these kinases to catalyze phosphotransfer from DivJ and PleC. We have used a yeast two-hybrid screen to investigate the molecular basis of DivJ and PleC interaction with DivK and to identify other His-Asp signal transduction proteins that interact with DivK. The only His-Asp proteins identified in the two-hybrid screen were five members of the histidine kinase superfamily. The finding that most of the kinase clones isolated correspond to either DivJ or PleC supports the previous conclusion that DivJ and PleC are cognate DivK kinases. A 66-amino-acid sequence common to all cloned DivJ and PleC fragments contains the conserved helix 1, helix 2 sequence that forms a four-helix bundle in histidine kinases required for dimerization, autophosphorylation and phosphotransfer. We present results that indicate that the four-helix bundle subdomain is not only necessary for binding of the response regulator but also sufficient for in vivo recognition specificity between DivK and its cognate histidine kinases. The other three kinases identified in this study correspond to DivL, an essential tyrosine kinase belonging to the same kinase subfamily as DivJ and PleC, and the two previously uncharacterized, soluble histidine kinases CckN and CckO. We discuss the significance of these results as they relate to kinase response regulator recognition specificity and the fidelity of phosphotransfer in signal transduction pathways.

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HdaB: a novel and conserved DnaA-related protein that targets the RIDA process to stimulate replication initiation

Nucleic Acids Research ◽

10.1093/nar/gkz1193 ◽

2019 ◽

Vol 48 (5) ◽

pp. 2412-2423 ◽

Cited By ~ 1

Author(s):

Antonio Frandi ◽

Justine Collier

Keyword(s):

Stationary Phase ◽

Caulobacter Crescentus ◽

Replication Initiation ◽

Lag Phase ◽

Related Protein ◽

Free Living ◽

Changing Environments ◽

A Cell ◽

The Impact

Abstract Exquisite control of the DnaA initiator is critical to ensure that bacteria initiate chromosome replication in a cell cycle-coordinated manner. In many bacteria, the DnaA-related and replisome-associated Hda/HdaA protein interacts with DnaA to trigger the Regulatory Inactivation of DnaA (RIDA) and prevent over-initiation events. In the Caulobacter crescentus Alphaproteobacterium, the RIDA process also targets DnaA for its rapid proteolysis by Lon. The impact of the RIDA process on adaptation of bacteria to changing environments remains unexplored. Here, we identify a novel and conserved DnaA-related protein, named HdaB, and show that homologs from three different Alphaproteobacteria can inhibit the RIDA process, leading to over-initiation and cell death when expressed in actively growing C. crescentus cells. We further show that HdaB interacts with HdaA in vivo, most likely titrating HdaA away from DnaA. Strikingly, we find that HdaB accumulates mainly during stationary phase and that it shortens the lag phase upon exit from stationary phase. Altogether, these findings suggest that expression of hdaB during stationary phase prepares cells to restart the replication of their chromosome as soon as conditions improve, a situation often met by free-living or facultative intracellular Alphaproteobacteria.

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Bacterioferritin ofMagnetospirillum gryphiswaldenseIs a Heterotetraeicosameric Complex Composed of Functionally Distinct Subunits but Is Not Involved in Magnetite Biomineralization

mBio ◽

10.1128/mbio.02795-18 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 4

Author(s):

René Uebe ◽

Frederik Ahrens ◽

Jörg Stang ◽

Katharina Jäger ◽

Lars H. Böttger ◽

...

Keyword(s):

Oxidative Stress ◽

Magnetotactic Bacteria ◽

Functional Differentiation ◽

Spectroscopic Techniques ◽

Content Type ◽

Magnetospirillum Gryphiswaldense ◽

Ferroxidase Activity ◽

Magnetite Biomineralization ◽

Field Lines

ABSTRACTThe biomineralization pathway of magnetite in magnetotactic bacteria is still poorly understood and a matter of intense debates. In particular, the existence, nature, and location of possible mineral precursors of magnetite are not clear. One possible precursor has been suggested to be ferritin-bound ferrihydrite. To clarify its role for magnetite biomineralization, we analyzed and characterized ferritin-like proteins from the magnetotactic alphaproteobacteriumMagnetospirillum gryphiswaldenseMSR-1, employing genetic, biochemical, and spectroscopic techniques. Transmission Mössbauer spectroscopy of the wild type (WT) and a bacterioferritin (bfr) deletion strain uncovered that the presence of ferrihydrite in cells is coupled to the presence of Bfr. However,bfranddpsdeletion mutants, encoding another ferritin-like protein, or even mutants with their codeletion had no impact on magnetite formation in MSR-1. Thus, ferritin-like proteins are not involved in magnetite biomineralization and Bfr-bound ferrihydrite is not a precursor of magnetite biosynthesis. Using transmission electron microscopy and bacterial two-hybrid and electrophoretic methods, we also show that MSR-1 Bfr is an atypical representative of the Bfr subfamily, as it forms tetraeicosameric complexes from two distinct subunits. Furthermore, our analyses revealed that these subunits are functionally divergent, with Bfr1 harboring a ferroxidase activity while only Bfr2 contributes to heme binding. Because of this functional differentiation and the poor formation of homooligomeric Bfr1 complexes, only heterooligomeric Bfr protects cells from oxidative stressin vivo.In summary, our results not only provide novel insights into the biomineralization of magnetite but also reveal the unique properties of so-far-uncharacterized heterooligomeric bacterioferritins.IMPORTANCEMagnetotactic bacteria likeMagnetospirillum gryphiswaldenseare able to orient along magnetic field lines due to the intracellular formation of magnetite nanoparticles. Biomineralization of magnetite has been suggested to require a yet-unknown ferritin-like ferrihydrite component. Here, we report the identification of a bacterioferritin as the source of ferrihydrite inM. gryphiswaldenseand show that, contrary to previous reports, bacterioferritin is not involved in magnetite biomineralization but required for oxidative stress resistance. Additionally, we show that bacterioferritin ofM. gryphiswaldenseis an unusual member of the bacterioferritin subfamily as it is composed of two functionally distinct subunits. Thus, our findings extend our understanding of the bacterioferritin subfamily and also solve a longstanding question about the magnetite biomineralization pathway.

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Two missense mutations identified in venous thrombosis patients impair the inhibitory function of the protein Z dependent protease inhibitor

Thrombosis and Haemostasis ◽

10.1160/th11-10-0708 ◽

2012 ◽

Vol 107 (05) ◽

pp. 854-863 ◽

Cited By ~ 6

Author(s):

Nigel P. Birch ◽

Peter J. Browett ◽

Paul B. Coughlin ◽

Anita J. Horvath ◽

Neil S. Van de Water ◽

...

Keyword(s):

Protease Inhibitor ◽

Venous Thrombosis ◽

Nonsense Mutation ◽

Healthy Controls ◽

Missense Mutations ◽

Wild Type ◽

Protein Z ◽

Inhibitory Function

SummaryProtein Z-dependent protease inhibitor (ZPI) is a plasma inhibitor of factor (F)Xa and FXIa. In an earlier study, five mutations were identified within the ZPI gene of venous thrombosis patients and healthy controls. Two of these were nonsense mutations and three were missense mutations in important regions of the protein. Here we report that two of these latter three mutations, F145L and Q384R, impair the inhibitory function of ZPI in vitro. Recombinant wild-type and mutant proteins were prepared; stability in response to thermal challenge was similar. Inhibition of FXa in the presence of the cofactor protein Z was reduced 68-fold by the Q384R mutant; inhibition of FXIa by the F145L mutant was reduced two- to three-fold compared to the wild-type ZPI. An analysis of all five ZPI mutations was undertaken in a cohort of venous thrombosis patients (n=550) compared to healthy controls (n=600). Overall, there was a modest increase in incidence of these mutations in the thrombosis group (odds ratio 2.0, 1.05–3.7, p=0.044). However, in contrast to W324X (nonsense mutation), the Q384R missense mutation and R88X nonsense mutation were evenly distributed in patients and controls; F145L was rare. The final mutation (S143Y) was also rare and did not significantly alter ZPI function in laboratory studies. The F145L and particularly the Q384R mutation impaired the function of the coagulation inhibitor ZPI; however, there was no convincing association between these mutations and venous thrombosis risk. The functional role for ZPI in vivo has yet to be clarified.

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Caulobacterrequires a dedicated mechanism to initiate chromosome segregation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0807448105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15435-15440 ◽

Cited By ~ 100

Author(s):

Esteban Toro ◽

Sun-Hae Hong ◽

Harley H. McAdams ◽

Lucy Shapiro

Keyword(s):

Chromosome Segregation ◽

Caulobacter Crescentus ◽

Time Lapse ◽

Replication Initiation ◽

Circular Chromosome ◽

Chromosomal Inversions ◽

Initiation Of Replication ◽

Time Lapse Imaging ◽

Single Circular Chromosome

Chromosome segregation in bacteria is rapid and directed, but the mechanisms responsible for this movement are still unclear. We show thatCaulobacter crescentusmakes use of and requires a dedicated mechanism to initiate chromosome segregation.Caulobacterhas a single circular chromosome whose origin of replication is positioned at one cell pole. Upon initiation of replication, an 8-kb region of the chromosome containing both the origin andparSmoves rapidly to the opposite pole. This movement requires the highly conservedParABSlocus that is essential inCaulobacter.We use chromosomal inversions andin vivotime-lapse imaging to show thatparSis theCaulobactersite of force exertion, independent of its position in the chromosome. WhenparSis moved farther from the origin, the cell waits forparSto be replicated before segregation can begin. Also, a mutation in the ATPase domain of ParA halts segregation without affecting replication initiation. Chromosome segregation inCaulobactercannot occur unless a dedicatedparSguiding mechanism initiates movement.

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HLA-A2-Restricted Protection against Lethal Lymphocytic Choriomeningitis

Journal of Virology ◽

10.1128/jvi.02063-06 ◽

2006 ◽

Vol 81 (5) ◽

pp. 2307-2317 ◽

Cited By ~ 16

Author(s):

Jason Botten ◽

J. Lindsay Whitton ◽

Polly Barrowman ◽

John Sidney ◽

Jason K. Whitmire ◽

...

Keyword(s):

T Cells ◽

Transgenic Mice ◽

Lymphocytic Choriomeningitis ◽

Target Cells ◽

Primary Role ◽

Protein Z ◽

Glycoprotein Precursor ◽

Antigen Presenting ◽

Lcmv Infection

ABSTRACT The consequences of human lymphocytic choriomeningitis virus (LCMV) infection can be severe, including aseptic meningitis in immunocompetent individuals, hydrocephalus or chorioretinitis in fetal infection, or a highly lethal outcome in immunosuppressed individuals. In murine models of LCMV infection, CD8+ T cells play a primary role in providing protective immunity, and there is evidence that cellular immunity may also be important in related arenavirus infections in humans. For this reason, we sought to identify HLA-A2 supertype-restricted epitopes from the LCMV proteome and evaluate them as vaccine determinants in HLA transgenic mice. We identified four HLA-A*0201-restricted peptides—nucleoprotein NP69-77, glycoprotein precursor GPC10-18, GPC447-455, and zinc-binding protein Z49-58—that displayed high-affinity binding (≤275 nM) to HLA-A*0201, induced CD8+ T-cell responses of high functional avidity in HLA-A*0201 transgenic mice, and were naturally processed from native LCMV antigens in HLA-restricted human antigen presenting cells. One of the epitopes (GPC447-455), after peptide immunization of HLA-A*0201 mice, induced CD8+ T cells capable of killing peptide-pulsed HLA-A*0201-restricted target cells in vivo and protected mice against lethal intracranial challenge with LCMV.

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