scholarly journals Hampered motility promotes the evolution of wrinkly phenotype in Bacillus subtilis

2018 ◽  
Author(s):  
Anne Richter ◽  
Theresa Hölscher ◽  
Patrick Pausch ◽  
Tim Sehrt ◽  
Franziska Brockhaus ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Dna Interaction ◽  
Bacterial Cells ◽  
Wild Type ◽  
Motile Cells ◽  
The Matrix ◽  
In The Wild ◽  
Selection For ◽  
Air Liquid Interface ◽  
Dna Binding Properties

SummarySelection for a certain trait in microbes depends on the genetic background of the strain and the selection pressure of the environmental conditions acting on the cells. In contrast to the sessile state in the biofilm, various bacterial cells employ flagellum-dependent motility under planktonic conditions suggesting that the two phenotypes are mutually exclusive. However, flagellum dependent motility facilitates the prompt establishment of floating biofilms on the air-medium interface, called pellicles. Previously, pellicles of B. subtilis were shown to be preferably established by motile cells, causing a reduced fitness of non-motile derivatives in the presence of the wild type strain. Here, we show that lack of fully assembled flagella promotes the evolution of matrix overproducers that can be distinguished by the characteristic wrinkled colony morphotype. The wrinkly phenotype is associated with amino acid substitutions in the master repressor of biofilm-related genes, SinR. By analyzing one of the mutations, we show that it alters the tetramerization and DNA binding properties of SinR, allowing an increased expression of the operon responsible for exopolysaccharide production. Finally, we demonstrate that the wrinkly phenotype is advantageous when cells lack flagella, but not in the wild type background.Graphical AbstractAbbreviated SummaryDuring biofilm establishment at the air-liquid interface, Bacillus subtilis evolves matrix overproducers with a wrinkly colony phenotype (WS). This is caused by mutations in the regulator SinR which alter its dimerization and DNA interaction properties. The matrix overproducers appear mostly in a non-motile mutant where they possess a competitive advantage for biofilm formation, which is not present in the wild type background.

scholarly journals Effects of oriC relocation on control of replication initiation in Bacillus subtilis

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3070-3082 ◽  
Author(s):  
Shigeki Moriya ◽  
Yoshikazu Kawai ◽  
Sakiko Kaji ◽  
Adrian Smith ◽  
Elizabeth J. Harry ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bacillus Subtilis ◽  
Dna Replication ◽  
Negative Control ◽  
Replication Initiation ◽  
Control Mechanisms ◽  
Bacterial Cells ◽  
Wild Type ◽  
Dna Replication Initiation ◽  
In The Wild

In bacteria, DNA replication initiation is tightly regulated in order to coordinate chromosome replication with cell growth. In Escherichia coli, positive factors and negative regulatory mechanisms playing important roles in the strict control of DNA replication initiation have been reported. However, it remains unclear how bacterial cells recognize the right time for replication initiation during the cell cycle. In the Gram-positive bacterium Bacillus subtilis, much less is known about the regulation of replication initiation, specifically, regarding negative control mechanisms which ensure replication initiation only once per cell cycle. Here we report that replication initiation was greatly enhanced in strains that had the origin of replication (oriC) relocated to various loci on the chromosome. When oriC was relocated to new loci further than 250 kb counterclockwise from the native locus, replication initiation became asynchronous and earlier than in the wild-type cells. In two oriC-relocated strains (oriC at argG or pnbA, 25 ° or 30 ° on the 36 ° chromosome map, respectively), DnaA levels were higher than in the wild-type but not enough to cause earlier initiation of replication. Our results suggest that the initiation capacity of replication is accumulated well before the actual time of initiation, and its release may be suppressed by a unique DNA structure formed near the native oriC locus.

scholarly journals Analysis of epitope distribution of arabinogalactan protein-extensins in pea (Pisum Sativum) nodules of wild-type and mutants impaired in infection thread growth

2019 ◽  
Vol 17 (3) ◽  
pp. 5-12
Author(s):  
Anna V. Tsyganova ◽  
Nicholas J. Brewin ◽  
Viktor E. Tsyganov
Keyword(s):  
Mutant Line ◽  
Arabinogalactan Protein ◽  
Infection Thread ◽  
Immunogold Electron Microscopy ◽  
Bacterial Cells ◽  
Wild Type ◽  
Infection Threads ◽  
The Matrix ◽  
Pea Mutants ◽  
High Level

Background. During the colonization of root and nodule tissues of legumes by rhizobia, bacterial cells are immersed in a plant extracellular matrix which includes arabinogalactan protein-extensins (AGPE). Materials and methods. Immunogold electron microscopy with monoclonal antibodies MAC204 and MAC236 was used to analyse the distribution and abundance of epitopes of AGPE in wild-type and symbiotically defective pea mutants. Results. In the nodules of the wild-type line SGE, both AGPE epitopes were detected to the same extent in the matrix of infection threads and infection droplets. In the nodules of the mutant line SGEFix-1 (sym40), the level of labelling by MAC204 was significantly higher than with SGE in both infection threads and infection droplets, but the level of labelling by MAC236 was only increased in the infection droplets. In the mutant line SGEFix-2 (sym33-3), a relatively high level of both epitopes was observed among all analysed genotypes. The double mutant line RBT3 (sym33-3, sym40) showed an intermediate level of labelling for both epitopes in infection threads compared with the parental mutants. In SGEFix-1, an abnormal distribution of both epitopes was observed in the intercellular space matrix. The MAC204 epitope was found in the cell walls of SGEFix-1 and in the infection thread walls of SGEFix-2, whereas in RBT3 this epitope was detected in both types of walls. Conclusions. The sym33-3 and sym40 mutations have different effects on the accumulation of AGPE epitopes recognised by MAC204 and MAC236. This indicates that both the Sym33 and the Sym40 genes affect the composition of AGPE in the matrix of infection threads and infection droplets.

scholarly journals Both galactosaminogalactan and α-1,3-glucan contribute to aggregation of Aspergillus oryzae hyphae in liquid culture

2019 ◽  
Author(s):  
Ken Miyazawa ◽  
Akira Yoshimi ◽  
Motoaki Sano ◽  
Fuka Tabata ◽  
Asumi Sugahara ◽  
...  
Keyword(s):  
Filamentous Fungi ◽  
Aspergillus Oryzae ◽  
Double Mutant ◽  
Liquid Culture ◽  
Bacterial Cells ◽  
Wild Type ◽  
Hydrogen Bond Formation ◽  
Relative Contribution ◽  
In The Wild ◽  
Increase Productivity

AbstractFilamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.ImportanceProduction using filamentous fungi is an important part of the fermentation industry, but hyphal aggregation in these fungi in liquid culture limits productivity compared with that of yeast or bacterial cells. We found that galactosaminogalactan and α-1,3-glucan both function in hyphal aggregation in Aspergillus oryzae, and that the hyphae of a double mutant deficient in both polysaccharides become fully dispersed in liquid culture. We also revealed the relative contribution of α-1,3-glucan and galactosaminogalactan to hyphal aggregation. Recombinant protein production was higher in the double mutant than in the wild-type strain. Our research provides a potential technical innovation for the fermentation industry that uses filamentous fungi, as regulation of the growth characteristics of A. oryzae in liquid culture may increase productivity.

scholarly journals Decay of ermC mRNA in a Polynucleotide Phosphorylase Mutant of Bacillus subtilis

1998 ◽  
Vol 180 (22) ◽  
pp. 5968-5977 ◽  
Author(s):  
David H. Bechhofer ◽  
Wei Wang
Keyword(s):  
Bacillus Subtilis ◽  
Rna Structure ◽  
Northern Blot Analysis ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Gene Coding ◽  
In The Wild ◽  
Rna Fragments ◽  
Specific Mrna

ABSTRACT ermC mRNA decay was examined in a mutant ofBacillus subtilis that has a deleted pnpA gene (coding for polynucleotide phosphorylase). 5′-proximal RNA fragments less than 400 nucleotides in length were abundant in thepnpA strain but barely detectable in the wild type. On the other hand, the patterns of 3′-proximal RNA fragments were similar in the wild-type and pnpA strains. Northern blot analysis with different probes showed that the 5′ end of the decay intermediates was the native ermC 5′ end. For one prominent ermCRNA fragment, in particular, it was shown that formation of its 3′ end was directly related to the presence of a stalled ribosome. 5′-proximal decay intermediates were also detected for transcripts encoded by theyybF gene. These results suggest that PNPase activity, which may be less sensitive to structures or sequences that block exonucleolytic decay, is required for efficient decay of specific mRNA fragments. However, it was shown that even PNPase activity could be blocked in vivo at a particular RNA structure.

scholarly journals Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

2019 ◽  
Vol 85 (17) ◽  
Author(s):  
Zhu Jiang ◽  
Tengfei Niu ◽  
Xueqin Lv ◽  
Yanfeng Liu ◽  
Jianghua Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Efficiency ◽  
Molecular Engineering ◽  
Fine Tuning ◽  
Wild Type ◽  
Content Type ◽  
Chitosan Oligosaccharides ◽  
In The Wild ◽  
Secretory Production ◽  
Extracellular Activity

ABSTRACT Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5′ untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax increased from 5.11 μM s−1 in the wild type to 7.56 μM s−1 in the mutant R157T. IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.

scholarly journals Mutations in the lrpE Gene of Ralstonia solanacearum Affects Hrp Pili Production and Virulence

2006 ◽  
Vol 19 (8) ◽  
pp. 884-895 ◽  
Author(s):  
Yukio Murata ◽  
Naoyuki Tamura ◽  
Kazuhiro Nakaho ◽  
Takafumi Mukaihara
Keyword(s):  
Cell Surface ◽  
Growth Phase ◽  
Ralstonia Solanacearum ◽  
Vascular System ◽  
Exponential Growth Phase ◽  
Bacterial Cells ◽  
Wild Type ◽  
Bacterial Surface ◽  
Bacterial Morphology ◽  
In The Wild

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the ΔlrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the ΔlrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the ΔlrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.

scholarly journals Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis

2002 ◽  
Vol 184 (9) ◽  
pp. 2500-2520 ◽  
Author(s):  
Christine Eymann ◽  
Georg Homuth ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Profile Analysis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Dependent Manner ◽  
Wild Type ◽  
Stringent Control ◽  
In The Wild ◽  
Growth And Reproduction

ABSTRACT The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to dl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

scholarly journals Coiled-coil registry shifts in the F684I mutant of Bicaudal result in cargo-independent activation of dynein motility

2019 ◽  
Author(s):  
Heying Cui ◽  
Kathleen M. Trybus ◽  
M. Yusuf Ali ◽  
Puja Goyal ◽  
Kaiqi Zhang ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Underlying Mechanism ◽  
Structural Flexibility ◽  
Human Homolog ◽  
Wild Type ◽  
Transport Pathways ◽  
X Ray ◽  
In The Wild ◽  
Selection For ◽  
Dependent Transport

ABSTRACTThe dynein adaptor Drosophila Bicaudal D (BicD) is auto-inhibited and activates dynein motility only after cargo is bound, but the underlying mechanism is elusive. In contrast, we show that the full-length BicD/F684I mutant activates dynein processivity even in the absence of cargo. Our X-ray structure of the C-terminal domain of the BicD/F684I mutant reveals a coiled-coil registry shift; in the N-terminal region, the two helices of the homodimer are aligned, whereas they are vertically shifted in the wild-type. One chain is partially disordered and this structural flexibility is confirmed by computations, which reveal that the mutant transitions back and forth between the two registries. We propose that a coiled-coil registry shift upon cargo binding activates BicD for dynein recruitment. Moreover, the human homolog BicD2/F743I exhibits diminished binding of cargo adaptor Nup358, implying that a coiled-coil registry shift may be a mechanism to modulate cargo selection for BicD2–dependent transport pathways.

scholarly journals Involvement of Stringent Factor RelA in Expression of the Alkaline Protease Gene aprE in Bacillus subtilis

2001 ◽  
Vol 183 (15) ◽  
pp. 4648-4651 ◽  
Author(s):  
Michihiro Hata ◽  
Mitsuo Ogura ◽  
Teruo Tanaka
Keyword(s):  
Bacillus Subtilis ◽  
Alkaline Protease ◽  
Double Mutant ◽  
Regulatory System ◽  
Protease Gene ◽  
Wild Type ◽  
Double Mutation ◽  
Two Component ◽  
In The Wild ◽  
Alkaline Protease Gene

ABSTRACT Expression of Bacillus subtilis aprE, encoding an extracellular alkaline protease, is positively regulated by phosphorylated DegU, the regulator of a two-component regulatory system, DegS-DegU. We found that the expression of anaprE′-′lacZ fusion was greatly reduced in a disruption mutant with a mutation of relA, which encodes the stringent factor RelA. The level of DegU in the relA mutant was similar to that in the wild-type cell. A relA degU double mutation did not result in a further decrease of theaprE′-′lacZ level found in a degU single mutant. The expression of the aprE′-′lacZ fusion in therelA mutant was stimulated by multicopy degR or the degU32(Hy) and degS200(Hy) mutations that cause the stabilization of phosphorylated DegU. Furthermore, the expression of sacB′-′lacZ, which is also dependent on phosphorylated DegU, was stimulated by the relA mutation, and this stimulation was not seen in the relA degU double mutant. These results show that RelA (or its product guanosine-3′,5′-bisdiphosphate [pp Gpp]) does not affect the phosphorylation of DegU and suggest that it participates in the expression of aprE and sacB through the regulation of DegU-dependent transcription.

scholarly journals Binding of σA and σB to Core RNA Polymerase after Environmental Stress in Bacillus subtilis

2003 ◽  
Vol 185 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Claudia Rollenhagen ◽  
Haike Antelmann ◽  
Janine Kirstein ◽  
Olivier Delumeau ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Environmental Stress ◽  
Ethanol Stress ◽  
Alternative Sigma Factor ◽  
Wild Type ◽  
The Core ◽  
In The Wild ◽  
Rna Polymerase Holoenzyme ◽  
Stress Gene

ABSTRACT In Bacillus subtilis, the alternative sigma factor σB is activated in response to environmental stress or energy depletion. The general stress regulon under the control of σB provides the cell with multiple stress resistance. Experiments were designed to determine how activated σB replaces σA as a constituent of the RNA polymerase holoenzyme. Studies of the transcription of the σA-dependent stress gene clpE under σB-inducing conditions showed that expression was higher in a sigB mutant background than in the wild type. The relative affinities of σA and σB for binding to the core RNA polymerase (E) were determined by means of indirect surface plasmon resonance. The results showed that the affinity of σB for E was 60-fold lower than that of σA. Western blot analyses with antibodies against σA, σB, and E showed that, after exposure to ethanol stress, the concentration of σB was only twofold higher than those of σA and E. Thus, the concentration of σB after stress is not high enough to compensate for its relatively low affinity for E, and it seems that additional mechanisms must be invoked to account for the binding of σB to E after stress.

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