scholarly journals Requirement of the LtsA Protein for Formation of the Mycolic Acid-Containing Layer on the Cell Surface of Corynebacterium glutamicum

2021 ◽  
Vol 9 (2) ◽  
pp. 409
Author(s):  
Yutaro Kumagai ◽  
Takashi Hirasawa ◽  
Masaaki Wachi
Keyword(s):  
Cell Division ◽  
Corynebacterium Glutamicum ◽  
Fluorescent Dyes ◽  
Intracellular Localization ◽  
Mycolic Acid ◽  
Differential Staining ◽  
Wild Type ◽  
Terminal Domain ◽  
Mutant Cells ◽  
Increased Susceptibility

The ltsA gene of Corynebacterium glutamicum encodes a purF-type glutamine-dependent amidotransferase, and mutations in this gene result in increased susceptibility to lysozyme. Recently, it was shown that the LtsA protein catalyzes the amidation of diaminopimelate residues in the lipid intermediates of peptidoglycan biosynthesis. In this study, intracellular localization of wild-type and mutant LtsA proteins fused with green fluorescent protein (GFP) was investigated. The GFP-fused wild-type LtsA protein showed a peripheral localization pattern characteristic of membrane-associated proteins. The GFP-fusions with a mutation in the N-terminal domain of LtsA, which is necessary for the glutamine amido transfer reaction, exhibited a similar localization to the wild type, whereas those with a mutation or a truncation in the C-terminal domain, which is not conserved among the purF-type glutamine-dependent amidotransferases, did not. These results suggest that the C-terminal domain is required for peripheral localization. Differential staining of cell wall structures with fluorescent dyes revealed that formation of the mycolic acid-containing layer at the cell division planes was affected in the ltsA mutant cells. This was also confirmed by observation that bulge formation was induced at the cell division planes in the ltsA mutant cells upon lysozyme treatment. These results suggest that the LtsA protein function is required for the formation of a mycolic acid-containing layer at the cell division planes and that this impairment results in increased susceptibility to lysozyme.

scholarly journals Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

2000 ◽  
Vol 182 (14) ◽  
pp. 3965-3971 ◽  
Author(s):  
Zonglin Hu ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Functional Domains ◽  
Wild Type ◽  
Terminal Domain ◽  
Ring Assembly ◽  
Z Ring ◽  
Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

scholarly journals Identification and Functional Analysis of the Gene Cluster for l-Arabinose Utilization in Corynebacterium glutamicum

2009 ◽  
Vol 75 (11) ◽  
pp. 3419-3429 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Miho Sasaki ◽  
Alain A. Vertès ◽  
Masayuki Inui ◽  
Hideaki Yukawa
Keyword(s):  
Growth Rate ◽  
Corynebacterium Glutamicum ◽  
Gene Cluster ◽  
Wild Type Strain ◽  
Transcriptional Regulator ◽  
Transcriptional Unit ◽  
Gene Products ◽  
Wild Type ◽  
Mutant Cells ◽  
Arabinose Isomerase

ABSTRACT Corynebacterium glutamicum ATCC 31831 grew on l-arabinose as the sole carbon source at a specific growth rate that was twice that on d-glucose. The gene cluster responsible for l-arabinose utilization comprised a six-cistron transcriptional unit with a total length of 7.8 kb. Three l-arabinose-catabolizing genes, araA (encoding l-arabinose isomerase), araB (l-ribulokinase), and araD (l-ribulose-5-phosphate 4-epimerase), comprised the araBDA operon, upstream of which three other genes, araR (LacI-type transcriptional regulator), araE (l-arabinose transporter), and galM (putative aldose 1-epimerase), were present in the opposite direction. Inactivation of the araA, araB, or araD gene eliminated growth on l-arabinose, and each of the gene products was functionally homologous to its Escherichia coli counterpart. Moreover, compared to the wild-type strain, an araE disruptant exhibited a >80% decrease in the growth rate at a lower concentration of l-arabinose (3.6 g liter−1) but not at a higher concentration of l-arabinose (40 g liter−1). The expression of the araBDA operon and the araE gene was l-arabinose inducible and negatively regulated by the transcriptional regulator AraR. Disruption of araR eliminated the repression in the absence of l-arabinose. Expression of the regulon was not repressed by d-glucose, and simultaneous utilization of l-arabinose and d-glucose was observed in aerobically growing wild-type and araR deletion mutant cells. The regulatory mechanism of the l-arabinose regulon is, therefore, distinct from the carbon catabolite repression mechanism in other bacteria.

scholarly journals DivIVA is essential in Deinococcus radiodurans and its C terminal domain regulates new septum orientation during cell division

2020 ◽  
Author(s):  
Reema Chaudhary ◽  
Swathi Kota ◽  
Hari S Misra
Keyword(s):  
Cell Division ◽  
Deinococcus Radiodurans ◽  
Wild Type ◽  
Orthogonal Axis ◽  
Septum Formation ◽  
Terminal Domain ◽  
Cell Position ◽  
Type Pattern ◽  
In Trans ◽  
Ftsz Assembly

AbstractFtsZ assembly at mid cell position in rod shaped bacteria is regulated by gradient of MinCDE complex across the poles. In round shaped bacteria, which lack predefined poles and the next plane of cell division is perpendicular to previous plane, the determination of site for FtsZ assembly is intriguing. Deinococcus radiodurans a coccus shaped bacterium, is characterized for its extraordinary resistance to DNA damage. Here we report that DivIVA a putative component of Min system in this bacterium (drDivIVA) interacts with cognate cell division and genome segregation proteins. The deletion of full length drDivIVA was found to be indispensable while its C-terminal deletion (ΔdivIVAC) was dispensable but produced distinguishable phenotypes like slow growth, altered plane for new septum formation and angular septum. Both wild type and mutant showed FtsZ foci formation and their gamma radiation responses were nearly identical. But unlike in wild type, the FtsZ localization in mutant cells was found to be away from orthogonal axis with respect to plane of previous septum. Notably, DivIVA-RFP localizes to membrane during cell division and then perpendicular to previous plane of cell division. In trans expression of drDivIVA in ΔdivIVAC background could restore the wild type pattern of septum formation perpendicular to previous septum. These results suggested that DivIVA is an essential protein in D. radiodurans and the C-terminal domain that contributes to its interaction with MinC determines the plane of new septum formation, possibly by controlling MinC oscillation through orthogonal axis in the cells.

scholarly journals Nucleosomal association and altered interactome underlie the mechanism of cataract caused by the R54C mutation of αA-crystallin

2020 ◽  
Author(s):  
Saad M. Ahsan ◽  
Bakthisaran Raman ◽  
Tangirala Ramakrishna ◽  
Ch. Mohan Rao
Keyword(s):  
Molecular Basis ◽  
Quaternary Structure ◽  
Nuclear Translocation ◽  
Intracellular Localization ◽  
Small Heat Shock Protein ◽  
Chaperone Activity ◽  
Wild Type ◽  
Terminal Domain ◽  
Αb Crystallin ◽  
Arginine Residues

AbstractThe small heat shock protein (sHSP), αA-crystallin, plays an important role in eye lens development. It has three distinct domains viz. the N-terminal domain, α-crystallin domain and the C-terminal extension. While the α-crystallin domain is conserved across the sHSP family, the N-terminal domain and the C-terminal extension are comparatively less conserved. Nevertheless, certain arginine residues in the N-terminal region of αA-crystallin are conserved across the sHSP family. Interestingly, most of the cataractcausing mutations in αA-crystallin occur in the conserved arginine residues. In order to understand the molecular basis of cataract caused by the R54C mutation in human αA-crystallin, we have compared the structure, chaperone activity, intracellular localization, effect on cell viability and “interactome” of wild-type and mutant αA-crystallin. Although R54CαA-crystallin exhibited slight changes in quaternary structure, its chaperone activity was comparable to that of the wild-type. When expressed in lens epithelial cells, R54CαA-crystallin triggered a stress-like response, resulting in nuclear translocation of αB-crystallin, disassembly of cytoskeletal elements and activation of Caspase 3, leading to apoptosis. Comparison of the “interactome” of the wild-type and mutant proteins revealed a striking increase in the interaction of the mutant protein with nucleosomal histones (H2A, H2B, H3 and H4). Using purified chromatin fractions, we show an increased association of R54CαA-crystallin with these nucleosomal histones, suggesting the potential role of the mutant in transcriptional modulation. Thus, the present study shows that alteration of “interactome” and its nucleosomal association, rather than loss of chaperone activity, is the molecular basis of cataract caused by the R54C mutation in αA-crystallin.

scholarly journals Cell Structure Changes in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus Lacking the S-Layer

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Changyi Zhang ◽  
Rebecca L. Wipfler ◽  
Yuan Li ◽  
Zhiyu Wang ◽  
Emily N. Hallett ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Size ◽  
Cell Fusion ◽  
Cell Biology ◽  
Cell Structure ◽  
Evolutionary Relationship ◽  
Wild Type ◽  
Sulfolobus Islandicus ◽  
Mutant Cells

ABSTRACT Rediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to the understanding of archaeal cells is the surface layer (S-layer), which is commonly found in Archaea but whose in vivo function is unknown. Here, we investigate the architecture and cellular roles of the S-layer in the hyperthermophilic crenarchaeon Sulfolobus islandicus. Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact or partially or completely detached SlaA are observed for the ΔslaB mutant. We experimentally identify a novel S-layer-associated protein, M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Mutants deficient in the SlaA outer layer form large cell aggregates, and individual cell size varies, increasing significantly up to six times the diameter of wild-type cells. We show that the ΔslaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. The ΔslaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells, in which the cell cycle is tightly regulated. Together, these data suggest that the lack of SlaA results in either cell fusion or irregularities in cell division. Our studies show the key physiological and cellular functions of the S-layer in this archaeal cell. IMPORTANCE The S-layer is considered to be the sole component of the cell wall in Sulfolobales, a taxonomic group within the Crenarchaeota whose cellular features have been suggested to have a close relationship to the last archaea-eukaryote common ancestor. In this study, we genetically dissect how the two previously characterized S-layer genes as well as a newly identified S-layer-associated protein-encoding gene contribute to the S-layer architecture in Sulfolobus. We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or/and cell fusion.

scholarly journals Novel Chromosome Organization Pattern in Actinomycetales—Overlapping Replication Cycles Combined with Diploidy

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Kati Böhm ◽  
Fabian Meyer ◽  
Agata Rhomberg ◽  
Jörn Kalinowski ◽  
Catriona Donovan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Corynebacterium Glutamicum ◽  
Model Organism ◽  
Growth Conditions ◽  
Mycolic Acid ◽  
Model Organisms ◽  
Cell Cycles ◽  
Slow Growing

ABSTRACT Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum. IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum, an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods. IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum, an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods.

scholarly journals Role of SpoVG in Asymmetric Septation inBacillus subtilis

1999 ◽  
Vol 181 (11) ◽  
pp. 3392-3401 ◽  
Author(s):  
Kiyoshi Matsuno ◽  
Abraham L. Sonenshein
Keyword(s):  
Cell Division ◽  
Signal Transduction Pathway ◽  
Negative Regulator ◽  
Stage I ◽  
Loss Of Function ◽  
Wild Type ◽  
Gene Coding ◽  
Additional Defect ◽  
Cell Division Inhibitor ◽  
Mutant Cells

ABSTRACT Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and otherspoVG loss-of-function mutations enabled citCmutant cells to form asymmetric septa and to activate the forespore-specific sigma factor ςF. However, little induction of mother cell-specific, ςE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed ςF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

scholarly journals Microtubule-mitochondrial attachment determines cell division symmetry and polarity in fission yeast

2021 ◽  
Author(s):  
Leeba Ann Chacko ◽  
Vaishnavi Ananthanarayanan
Keyword(s):  
Cell Division ◽  
Fission Yeast ◽  
Daughter Cell ◽  
Significant Proportion ◽  
Intrinsic Factor ◽  
The Other ◽  
Wild Type ◽  
Feedback Controls ◽  
Unequal Distribution ◽  
Mutant Cells

Association with microtubules inhibits the fission of mitochondria in Schizosachharomyces pombe. Here we show that this attachment of mitochondria to microtubules is an important cell intrinsic factor in determining division symmetry as well as maintaining polarity. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. So too, microtubule pivoting was enhanced in both mitochondrial mutants, resulting in a fraction of the cells in these populations displaying polarity defects. The asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and thereby, cell growth.

scholarly journals The RipA and RipB Peptidoglycan Endopeptidases Are Individually Nonessential to Mycobacterium smegmatis

2016 ◽  
Vol 198 (9) ◽  
pp. 1464-1475 ◽  
Author(s):  
Daniel J. Martinelli ◽  
Martin S. Pavelka
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Mycobacterium Smegmatis ◽  
Wild Type ◽  
Content Type ◽  
Deletion Mutations ◽  
Levels Of Detail ◽  
Mutant Strains ◽  
Single Deletion ◽  
Increased Susceptibility

ABSTRACTMycobacteria possess a series of Rip peptidoglycan endopeptidases that have been characterized in various levels of detail. The RipA and RipB proteins have been extensively studied and aredl-endopeptidases, and RipA has been considered essential toMycobacterium smegmatisandMycobacterium tuberculosis. We show here that theripAandripBgenes are individually dispensable inM. smegmatisand that at least one of the genes must be expressed for viability. We characterized strains carrying in-frame deletion mutations ofripAandripBand found that both mutant strains exhibited increased susceptibility to a limited number of antibiotics and to detergent but that only the ΔripAmutant displayed hypersusceptibility to lysozyme. We also constructed and characterized ΔripDand ΔripAΔripDmutants and found that the single mutant had only an intermediate lysozyme hypersusceptibility phenotype compared to that of wild-type cells while loss ofripDin the ΔripAbackground partially rescued the antibiotic and lysozyme phenotypes of the ΔripAmutant.IMPORTANCEWe show that the RipA endopeptidase, which has been considered essential for cell division in certain mycobacteria, is not essential but that at least it or a similar protein, RipB, must be expressed by the bacteria for viability. This work is the first description of strains carrying single deletion mutations of RipA, RipB, and a novel endopeptidase-like protein, RipD.

scholarly journals Revealing S-layer Functions in the Hyperthermophilic Crenarchaeon Sulfolobus islandicus

2018 ◽  
Author(s):  
Changyi Zhang ◽  
Rebecca L. Wipfler ◽  
Yuan Li ◽  
Zhiyu Wang ◽  
Emily N. Hallett ◽  
...  
Keyword(s):  
Cell Division ◽  
Cell Size ◽  
Cell Fusion ◽  
Cell Biology ◽  
Cell Envelope ◽  
Evolutionary Relationship ◽  
Wild Type ◽  
Sulfolobus Islandicus ◽  
Mutant Cells

AbstractThe crystalline surface layer (S-layer), consisting of two glycoproteins SlaA and SlaB, is considered to be the exclusive component of the cell envelope outside of the cytoplasmic membrane in Sulfolobus species. Although biochemically and structurally characterized, the S-layer in vivo functions remain largely elusive in Archaea. Here, we investigate how the S-layer genes contribute to the S-layer architecture and affect cellular physiology in a crenarchaeal model, Sulfolobus islandicus M.16.4. Electron micrographs of mutant cells lacking slaA or both slaA and slaB confirm the absence of the outermost layer (SlaA), whereas cells with intact, partially, or completely detached SlaA are observed for the ∆slaB mutant. Importantly, we identify a novel S-layer-associated protein M164_1049, which does not functionally replace its homolog SlaB but likely assists SlaB to stabilize SlaA. Additionally, we find that mutants deficient in SlaA form large cell aggregates and the individual cell size varies significantly. The slaB gene deletion also causes noticeable cellular aggregation, but the size of those aggregates is smaller when compared to ∆slaA and ∆slaAB mutants. We further show the ∆slaA mutant cells exhibit more sensitivity to hyperosmotic stress but are not reduced to wild-type cell size. Finally, we demonstrate that the ∆slaA mutant contains aberrant chromosome copy numbers not seen in wild-type cells where the cell cycle is tightly regulated. Together these data suggest that the lack of slaA results in either cell fusion or irregularities in cell division. Our studies provide novel insights into the physiological and cellular functions of the S-layer in Archaea.SignificanceRediscovery of the ancient evolutionary relationship between archaea and eukaryotes has revitalized interest in archaeal cell biology. Key to understanding the archaeal cell is the S-layer which is ubiquitous in Archaea but whose in vivo function is unknown. In this study, we genetically dissect how the two well-known S-layer genes as well as a newly identified S-layer-associated-protein-encoding gene contribute to the S-layer architecture in a hyperthermophilic crenarchaeal model S. islandicus. We provide genetic evidence for the first time showing that the slaA gene is a key cell morphology determinant and may play a role in Sulfolobus cell division or cell fusion.

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