scholarly journals Regulatory Overlap and Functional Redundancy among Bacillus subtilis Extracytoplasmic Function σ Factors

2007 ◽  
Vol 189 (19) ◽  
pp. 6919-6927 ◽  
Author(s):  
Thorsten Mascher ◽  
Anna-Barbara Hachmann ◽  
John D. Helmann
Keyword(s):  
Mutant Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Physiological Role ◽  
Polymyxin B ◽  
Triple Mutant ◽  
Σ Factor ◽  
Quadruple Mutant ◽  
Phenotype Microarrays ◽  
Increased Sensitivity

ABSTRACT Bacillus subtilis encodes seven extracytoplasmic function (ECF) σ factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF σ factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF σ factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including β-lactams, polymyxin B, and d-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two σ factors.

scholarly journals Listeria monocytogenes 10403S Alternative Sigma-54 Factor σL Has a Negative Role on Survival Ability Under Bile Exposure

2021 ◽  
Vol 12 ◽  
Author(s):  
Atsadang Boonmee ◽  
Haley F. Oliver ◽  
Soraya Chaturongakul
Keyword(s):  
Listeria Monocytogenes ◽  
Triple Mutant ◽  
Σ Factor ◽  
Bile Resistance ◽  
Quadruple Mutant ◽  
Bile Fluid ◽  
Negative Role ◽  
Pass Through ◽  
Isogenic Mutants

Listeria monocytogenes is a Gram-positive bacterium causing listeriosis in animals and humans. To initiate a foodborne infection, L. monocytogenes has to pass through the host gastrointestinal tract (GIT). In this study, we evaluated survival abilities of L. monocytogenes 10403S wild type (WT) and its isogenic mutants in alternative sigma (σ) factor genes (i.e., sigB, sigC, sigH, and sigL) under simulated gastric, duodenal, and bile fluids. Within 10min of exposures, only bile fluid was able to significantly reduce survival ability of L. monocytogenes WT by 2 logs CFU/ml. Loss of sigL showed the greatest bile resistance among 16 strains tested, p<0.0001, (i.e., WT, four single alternative σ factor mutants, six double mutants, four triple mutants, and one quadruple mutant). To further investigate the role of σL in bile response, RNA-seq was conducted to compare the transcriptional profiles among L. monocytogenes 10403S ΔBCH triple mutant (lacking sigB, sigC, and sigH genes; expressing housekeeping σA and σL) and ΔBCHL quadruple mutant (lacking all alternative sigma factor genes; expressing only σA) strains under BHI and 1% bile conditions. A total of 216 and 176 differentially expressed genes (DEGs) were identified in BHI and bile, respectively. We confirmed that mpt operon was shown to be strongly activated by σL. Interestingly, more than 80% of DEGs were found to be negatively regulated in the presence of σL. This includes PrfA regulon and its mediated genes (i.e., hly, hpt, inlB, clpP, clpE, groL, and inlC) which were downregulated in response to bile in the presence of σL. This result suggests the potential negative role of σL on bile survival, and the roles of σL and σB might be in a seesaw model prior to host cell invasion.

scholarly journals Transcriptomic and Phenotypic Characterization of a Bacillus subtilis Strain without Extracytoplasmic Function σ Factors

2010 ◽  
Vol 192 (21) ◽  
pp. 5736-5745 ◽  
Author(s):  
Yun Luo ◽  
Kei Asai ◽  
Yoshito Sadaie ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Dominant Role ◽  
Phenotypic Characterization ◽  
Subtilis Strain ◽  
Intrinsic Resistance ◽  
Triple Mutant ◽  
Phenotypic Differences ◽  
Σ Factor

ABSTRACT Bacillus subtilis encodes seven extracytoplasmic function (ECF) σ factors. Three (σM, σW, and σX) mediate responses to cell envelope-active antibiotics. The functions of σV, σY, σZ, and σYlaC remain largely unknown, and strong inducers of these σ factors and their regulons have yet to be defined. Here, we define transcriptomic and phenotypic differences under nonstress conditions between a strain carrying deletions in all seven ECF σ factor genes (the Δ7ECF mutant), a ΔMWX triple mutant, and the parental 168 strain. Our results identify >80 genes as at least partially dependent on ECF σ factors, and as expected, most of these are dependent on σM, σW, or σX, which are active at a significant basal level during growth. Several genes, including the eps operon encoding enzymes for exopolysaccharide (EPS) production, were decreased in expression in the Δ7ECF mutant but affected less in the ΔMWX mutant. Consistent with this observation, the Δ7ECF mutant (but not the ΔMWX mutant) showed reduced biofilm formation. Extending previous observations, we also note that the ΔMWX mutant is sensitive to a variety of antibiotics and the Δ7ECF mutant is either as sensitive as, or slightly more sensitive than, the ΔMWX strain to these stressors. These findings emphasize the overlapping nature of the seven ECF σ factor regulons in B. subtilis, confirm that three of these (σM, σW, and σX) play the dominant role in conferring intrinsic resistance to antibiotics, and provide initial insights into the roles of the remaining ECF σ factors.

scholarly journals Bacillus subtilis σVConfers Lysozyme Resistance by Activation of Two Cell Wall Modification Pathways, Peptidoglycan O-Acetylation and d-Alanylation of Teichoic Acids

2011 ◽  
Vol 193 (22) ◽  
pp. 6223-6232 ◽  
Author(s):  
Veronica Guariglia-Oropeza ◽  
John D. Helmann
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Double Mutant ◽  
Cell Envelope ◽  
Intrinsic Resistance ◽  
Triple Mutant ◽  
Cell Wall Modification ◽  
Content Type ◽  
Teichoic Acids ◽  
Σ Factor

The seven extracytoplasmic function (ECF) sigma (σ) factors ofBacillus subtilisare broadly implicated in resistance to antibiotics and other cell envelope stressors mediated, in part, by regulation of cell envelope synthesis and modification enzymes. We here define the regulon of σVas including at least 20 operons, many of which are also regulated by σM, σX, or σW. The σVregulon is strongly and specifically induced by lysozyme, and this induction is key to the intrinsic resistance ofB. subtilisto lysozyme. Strains with null mutations in eithersigVor all seven ECF σ factor genes (Δ7ECF) have essentially equal increases in sensitivity to lysozyme. Induction of σVin the Δ7ECF background restores lysozyme resistance, whereas induction of σM, σX, or σWdoes not. Lysozyme resistance results from the ability of σVto activate the transcription of two operons: the autoregulatedsigV-rsiV-oatA-yrhKoperon anddltABCDE. Genetic analyses reveal thatoatAanddltare largely redundant with respect to lysozyme sensitivity: single mutants are not affected in lysozyme sensitivity, whereas anoatA dltAdouble mutant is as sensitive as asigVnull strain. Moreover, thesigV oatA dltAtriple mutant is no more sensitive than theoatA dltAdouble mutant, indicating that there are no other σV-dependent genes necessary for lysozyme resistance. Thus, we suggest that σVconfers lysozyme resistance by the activation of two cell wall modification pathways: O-acetylation of peptidoglycan catalyzed by OatA andd-alanylation of teichoic acids by DltABCDE.

scholarly journals Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

Microbiology ◽  
2010 ◽  
Vol 156 (11) ◽  
pp. 3492-3502 ◽  
Author(s):  
Nicole N. Driessen ◽  
Esther J. M. Stoop ◽  
Roy Ummels ◽  
Sudagur S. Gurcha ◽  
Arun K. Mishra ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Enzymic Activity ◽  
Mycobacterium Marinum ◽  
Phenotypic Traits ◽  
Wild Type ◽  
Exact Function ◽  
Acyl Chains

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM.

scholarly journals A Mutation of the RNA Polymerase β′ Subunit (rpoC) Confers Cephalosporin Resistance in Bacillus subtilis

2012 ◽  
Vol 57 (1) ◽  
pp. 56-65 ◽  
Author(s):  
Yong Heon Lee ◽  
Ki Hyun Nam ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Cell Envelope ◽  
Control Cell ◽  
Triple Mutant ◽  
Content Type ◽  
Σ Factor ◽  
Elevated Expression ◽  
Regulatory Effects ◽  
High Level

ABSTRACTIn bacteria, mutations affecting the major catalytic subunits of RNA polymerase (encoded byrpoBandrpoC) emerge in response to a variety of selective pressures. Here we isolated aBacillus subtilisstrain with high-level resistance to cefuroxime (CEF). Whole-genome resequencing revealed only one missense mutation affecting an invariant residue in close proximity to the C-terminal DNA-binding domain of RpoC (G1122D). Genetic reconstruction experiments demonstrate that this substitution is sufficient to confer CEF resistance. The G1122D mutation leads to elevated expression of stress-responsive regulons, including those of extracytoplasmic function (ECF) σ factors (σM, σW, and σX) and the general stress σ factor (σB). The increased CEF resistance of therpoCG1122Dstrain is lost in thesigM rpoCG1122Ddouble mutant, consistent with a major role for σMin CEF resistance. However, asigMmutant is very sensitive to CEF, and this sensitivity is still reduced by the G1122D mutation, suggesting that other regulatory effects are also important. Indeed, the ability of the G1122D mutation to increase CEF resistance is further reduced in a triple mutant strain lacking three ECF σ factors (σM, σW, and σX), which are known from prior studies to control overlapping sets of genes. Collectively, our findings highlight the ability of mutations in RNA polymerase to confer antibiotic resistance by affecting the activity of alternative σ factors that control cell envelope stress-responsive regulons.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals Establishing Virulence Associated Polyphosphate Kinase 2 as a drug target for Mycobacterium tuberculosis

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Mamta Singh ◽  
Prabhakar Tiwari ◽  
Garima Arora ◽  
Sakshi Agarwal ◽  
Saqib Kidwai ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Guinea Pigs ◽  
High Throughput Screening ◽  
Drug Target ◽  
Wild Type Strain ◽  
Wild Type ◽  
Inorganic Polyphosphate ◽  
Phosphate Donor ◽  
Microbial Stress

Abstract Inorganic polyphosphate (PolyP) plays an essential role in microbial stress adaptation, virulence and drug tolerance. The genome of Mycobacterium tuberculosis encodes for two polyphosphate kinases (PPK-1, Rv2984 and PPK-2, Rv3232c) and polyphosphatases (ppx-1, Rv0496 and ppx-2, Rv1026) for maintenance of intracellular PolyP levels. Microbial polyphosphate kinases constitute a molecular mechanism, whereby microorganisms utilize PolyP as phosphate donor for synthesis of ATP. In the present study we have constructed ppk-2 mutant strain of M. tuberculosis and demonstrate that PPK-2 enzyme contributes to its ability to cause disease in guinea pigs. We observed that ppk-2 mutant strain infected guinea pigs had significantly reduced bacterial loads and tissue pathology in comparison to wild type infected guinea pigs at later stages of infection. We also report that in comparison to the wild type strain, ppk-2 mutant strain was more tolerant to isoniazid and impaired for survival in THP-1 macrophages. In the present study we have standardized a luciferase based assay system to identify chemical scaffolds that are non-cytotoxic and inhibit M. tuberculosis PPK-2 enzyme. To the best of our knowledge this is the first study demonstrating feasibility of high throughput screening to obtain small molecule PPK-2 inhibitors.

scholarly journals Activation of 2,4-Diaminoquinazoline in Mycobacterium tuberculosis by Rv3161c, a Putative Dioxygenase

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
Eduard Melief ◽  
Shilah A. Bonnett ◽  
Edison S. Zuniga ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Type A ◽  
Wild Type ◽  
Metabolite Analysis ◽  
Content Type ◽  
Data Support ◽  
In The Wild

ABSTRACT The diaminoquinazoline series has good potency against Mycobacterium tuberculosis. Resistant isolates have mutations in Rv3161c, a putative dioxygenase. We carried out metabolite analysis on a wild-type strain and an Rv3161c mutant strain after exposure to a diaminoquinazoline. The parental compound was found in intracellular extracts from the mutant but not the wild type. A metabolite consistent with a monohydroxylated form was identified in the wild type. These data support the hypothesis that Rv3161c metabolizes diaminoquinazolines in M. tuberculosis.

Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

scholarly journals Mechanism of PQQ and CoQ10 overproduction in Methylobacterium as revealed by comparative genomic analysis between mutant and wild-type strains

2020 ◽  
Author(s):  
Changle Zhao ◽  
Yinping Wan ◽  
Xiaojie Cao ◽  
Huili Zhang ◽  
Xin Bao
Keyword(s):  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Comparative Genomic Analysis ◽  
Comparative Genomic ◽  
Regulation Mechanism ◽  
Whole Genome ◽  
Wild Type ◽  
Coq10 Production

Abstract Background The microbial synthesis of pyrroloquinoline quinone (PQQ) and Coenzyme Q10 (CoQ10) remains the most promising industrial production route. Methylobacterium has been used to generate PQQ and other value-added chemicals from cheap carbon feedstocks.However, the low PQQ and CoQ10 production capacity of the Methylobacterium strains is a major limitation The regulation mechanism for PQQ and CoQ10 biosynthesis in this strain has also not been fully elucidated. Results Methylobacterium sp. CLZ strain was isolated from soil contaminated with chemical wastewater, which can simultaneously produce PQQ, CoQ10, and carotenoids by using cheap methanol as carbon source. We investigated a mutant strain NI91, which increased the PQQ and CoQ10 yield by 72.44% and 59.80%, respectively. Whole-genome sequencing of NI91 and wild-type strain CLZ revealed that both contain a 5.28 Mb chromosome. The comparative genomic analysis and validation study revealed that a significant increase in biomass and PQQ production was associated with the base mutations in the methanol dehydrogenase (MDH) synthesis genes, mxaD and mxaJ. The significant increase in CoQ10 production may be associated with the base mutations in dxs gene, a key gene in the MEP/DOXP pathway. Conclusions A PQQ producing strain that simultaneously produces CoQ10 and carotenoids was selected and after ANI analysis, named as Methylobacterium sp. CLZ. After random mutagenesis of this strain, we obtained NI91 strain, which showed increased production of PQQ and CoQ10. Based on comparative genomic analysis of the whole genome of mutant strain NI91 and wild-type strain CLZ, a total of 270 SNPs and InDels events were detected, which provided a reference for subsequent research. The mutations in mxaD, mxaJ and dxs genes may be related to the high yield of PQQ and CoQ10. These findings will enhance our understanding of the PQQ and CoQ10 over-production mechanism in Methylobacterium sp. NI91 at the genomic level. It will also provide useful clues for strain engineering in order to improve the PQQ and CoQ10 production.

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