scholarly journals Two Types of Bacillus subtilis tetA(L) Deletion Strains Reveal the Physiological Importance of TetA(L) in K+ Acquisition as well as in Na+, Alkali, and Tetracycline Resistance

2000 ◽  
Vol 182 (8) ◽  
pp. 2088-2095 ◽  
Author(s):  
Wei Wang ◽  
Arthur A. Guffanti ◽  
Yi Wei ◽  
Masahiro Ito ◽  
Terry A. Krulwich
Keyword(s):  
Bacillus Subtilis ◽  
Tetracycline Resistance ◽  
Monovalent Cation ◽  
Alkali Resistance ◽  
Wild Type ◽  
Ph Homeostasis ◽  
Physiological Importance ◽  
Mutant Strains ◽  
In The Wild ◽  
Dependent Growth

ABSTRACT The chromosomally encoded TetA(L) protein of Bacillus subtilis is a multifunctional tetracycline-metal/H+antiporter that also exhibits monovalent cation/H+ antiport activity and a net K+ uptake mode. In this study, B. subtilis mutant strains JC112 and JC112C were found to be representative of two phenotypic types of tetA(L) deletion strains that are generated in the same selection. Both strains exhibited increased sensitivity to low tetracycline concentrations as expected. The mutants also had significantly reduced ability to grow in media containing low concentrations of K+, indicating that the net K+ uptake mode is of physiological consequence; the deficit in JC112 was greater than in JC112C. JC112 also exhibited (i) greater impairment of Na+- or K+-dependent growth at pH 8.3 than JC112C and (ii) a greater degree of Co+2 as well as Na+ sensitivity. Studies were initiated to explore the possibility of two different patterns of compensatory changes in other ion-translocating transporters in these mutants. Increased expression of two loci has thus far been shown. Increased expression of czcD-trkA, a locus with a proposed involvement in K+ uptake, occurred in both mutants. The increase was highest in the presence of Co2+ and was higher in JC112 than in JC112C. Deletion of czcD-trkA resulted in diminished growth of the wild-type and both mutant strains at low [K+], supporting a significant role for this locus in K+ uptake. Expression of yheL, which is a homologue of the Na+/H+ antiporter-encodingnhaC gene from Bacillus firmus OF4, was also increased in both tetA(L) deletion strains, again with higher up-regulation in JC112. The phenotypes resulting from deletion of yheL were consistent with a modest role for YheL in Na+-dependent pH homeostasis in the wild type. No major role for YheL was indicated in the mutants in spite of the overexpression. The studies underscore the multiple physiological functions of TetA(L), including tetracycline, Na+, and alkali resistance and K+ acquisition. The studies also reveal and begin to detail the complexity of the response to mutational loss of these functions.

scholarly journals Adaptive Gene Expression in Bacillus subtilis Strains Deleted for tetL

2006 ◽  
Vol 188 (20) ◽  
pp. 7090-7100 ◽  
Author(s):  
Yi Wei ◽  
Gintaras Deikus ◽  
Benjamin Powers ◽  
Victor Shelden ◽  
Terry A. Krulwich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Metal Ion ◽  
Expression Patterns ◽  
Tetracycline Resistance ◽  
Monovalent Cation ◽  
Deletion Strain ◽  
Wild Type ◽  
Genetic Changes ◽  
Citrate Transporter ◽  
Solute Uptake

ABSTRACT The growth properties of a new panel of Bacillus subtilis tetL deletion strains and of a derivative set of strains in which tetL is restored to the chromosome support earlier indications that deletion of tetL results in a range of phenotypes that are unrelated to tetracycline resistance. These phenotypes were not reversed by restoration of a tetL gene to its native locus and were hypothesized to result from secondary mutations that arise when multifunctional tetL is deleted. Such genetic changes would temper the alkali sensitivity and Na+ sensitivity that accompany loss of the monovalent cation/proton activity of TetL. Microarray comparisons of the transcriptomes of wild-type B. subtilis, a tetL deletion strain, and its tetL-restored derivative showed that 37 up-regulated genes and 13 down-regulated genes in the deletion strain did not change back to wild-type expression patterns after tetL was returned to the chromosome. Up-regulation of the citM gene, which encodes a divalent metal ion-coupled citrate transporter, was shown to account for the Co2+-sensitive phenotype of tetL mutants. The changes in expression of citM and genes encoding other ion-coupled solute transporters appear to be adaptive to loss of TetL functions in alkali and Na+ tolerance, because they reduce Na+-coupled solute uptake and enhance solute uptake that is coupled to H+ entry.

scholarly journals Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marianna Iorio ◽  
Sahar Davatgarbenam ◽  
Stefania Serina ◽  
Paolo Criscenzo ◽  
Mitja M. Zdouc ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Biosynthetic Gene Cluster ◽  
Systematic Investigation ◽  
Biosynthetic Gene ◽  
Wild Type ◽  
Bacterial Rna ◽  
Soil Isolate ◽  
Mutant Strains ◽  
In The Wild ◽  
Polymerase Inhibitor

AbstractWe report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

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 Transformation-deficient mutants of Bacillus subtilis impaired in competence-specific nuclease activities

1982 ◽  
Vol 152 (1) ◽  
pp. 166-174
Author(s):  
J A Mulder ◽  
G Venema
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Sodium Dodecyl Sulfate ◽  
Sodium Dodecyl ◽  
Magnesium Ions ◽  
Polyacrylamide Gels ◽  
Wild Type ◽  
Molecular Weights ◽  
Defective Mutant ◽  
Mutant Strains

A comparison of the nucleolytic activities in competent and physiologically low-competent wild-type cultures of Bacillus subtilis in DNA-containing sodium dodecyl sulfate-polyacrylamide gels revealed the existence of three competence-associated nuclease activities with apparent molecular weights of 13,000, 15,000, and 26,000. The three activities, which were dependent on manganese or magnesium ions, were specifically present in the competent fraction of a competent culture. The competence-associated nucleolytic activities of eight transformation-defective mutant strains were assayed, resulting in the following three classes of mutants: (i) four strains which, according to this assay, were not impaired in any of the nucleolytic activities mentioned above; (ii) one strain which was strongly impaired in the 13,000- and 26,000-molecular-weight activities, but showed a considerable level of the 15,000-molecular-weight activity; and (iii) three strains which were severely impaired in all three activities. The results indicated that the 26,000-molecular-weight activity was a dimer of the 13,000-molecular-weight activity and that this nuclease was involved in the entry of DNA.

scholarly journals Addition of Poly(A) and Heteropolymeric 3′ Ends in Bacillus subtilis Wild-Type and Polynucleotide Phosphorylase-Deficient Strains

2005 ◽  
Vol 187 (14) ◽  
pp. 4698-4706 ◽  
Author(s):  
Juan Campos-Guillén ◽  
Patricia Bralley ◽  
George H. Jones ◽  
David H. Bechhofer ◽  
Gabriela Olmedo-Alvarez
Keyword(s):  
Bacillus Subtilis ◽  
Bacterial Species ◽  
Synthetic Activity ◽  
Polynucleotide Phosphorylase ◽  
Wild Type ◽  
Mutant Strains ◽  
Mean Size ◽  
Identical Nucleotide ◽  
End Sequences ◽  
Polymerase I

ABSTRACT Polyadenylation plays a role in decay of some bacterial mRNAs, as well as in the quality control of stable RNA. In Escherichia coli, poly(A) polymerase I (PAP I) is the main polyadenylating enzyme, but the addition of 3′ tails also occurs in the absence of PAP I via the synthetic activity of polynucleotide phosphorylase (PNPase). The nature of 3′-tail addition in Bacillus subtilis, which lacks an identifiable PAP I homologue, was studied. Sizing of poly(A) sequences revealed a similar pattern in wild-type and PNPase-deficient strains. Sequencing of 152 cloned cDNAs, representing 3′-end sequences of nontranslated and translated RNAs, revealed modified ends mostly on incomplete transcripts, which are likely to be decay intermediates. The 3′-end additions consisted of either short poly(A) sequences or longer heteropolymeric ends with a mean size of about 40 nucleotides. Interestingly, multiple independent clones exhibited complex heteropolymeric ends of very similar but not identical nucleotide sequences. Similar polyadenylated and heteropolymeric ends were observed at 3′ ends of RNA isolated from wild-type and pnpA mutant strains. These data demonstrated that, unlike the case of some other bacterial species and chloroplasts, PNPase of Bacillus subtilis is not the major enzyme responsible for the addition of nucleotides to RNA 3′ ends.

scholarly journals Ohr plays a central role in bacterial responses against fatty acid hydroperoxides and peroxynitrite

2016 ◽  
Vol 114 (2) ◽  
pp. E132-E141 ◽  
Author(s):  
Thiago G. P. Alegria ◽  
Diogo A. Meireles ◽  
José R. R. Cussiol ◽  
Martín Hugo ◽  
Madia Trujillo ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Wild Type Strain ◽  
Wild Type ◽  
Glutathione Peroxidases ◽  
Mutant Strains ◽  
In The Wild ◽  
Acid Hydroperoxide ◽  
Lipoamide Dehydrogenase ◽  
Fatty Acid Hydroperoxides ◽  
Competition Assays

Organic hydroperoxide resistance (Ohr) enzymes are unique Cys-based, lipoyl-dependent peroxidases. Here, we investigated the involvement of Ohr in bacterial responses toward distinct hydroperoxides. In silico results indicated that fatty acid (but not cholesterol) hydroperoxides docked well into the active site of Ohr fromXylella fastidiosaand were efficiently reduced by the recombinant enzyme as assessed by a lipoamide-lipoamide dehydrogenase–coupled assay. Indeed, the rate constants between Ohr and several fatty acid hydroperoxides were in the 107–108M−1⋅s−1range as determined by a competition assay developed here. Reduction of peroxynitrite by Ohr was also determined to be in the order of 107M−1⋅s−1at pH 7.4 through two independent competition assays. A similar trend was observed when studying the sensitivities of a ∆ohrmutant ofPseudomonas aeruginosatoward different hydroperoxides. Fatty acid hydroperoxides, which are readily solubilized by bacterial surfactants, killed the ∆ohrstrain most efficiently. In contrast, both wild-type and mutant strains deficient for peroxiredoxins and glutathione peroxidases were equally sensitive to fatty acid hydroperoxides. Ohr also appeared to play a central role in the peroxynitrite response, because the ∆ohrmutant was more sensitive than wild type to 3-morpholinosydnonimine hydrochloride (SIN-1 , a peroxynitrite generator). In the case of H2O2insult, cells treated with 3-amino-1,2,4-triazole (a catalase inhibitor) were the most sensitive. Furthermore, fatty acid hydroperoxide and SIN-1 both induced Ohr expression in the wild-type strain. In conclusion, Ohr plays a central role in modulating the levels of fatty acid hydroperoxides and peroxynitrite, both of which are involved in host–pathogen interactions.

scholarly journals Effects of Nonpolar Mutations in Each of the Seven Bacillus subtilis mrp Genes Suggest Complex Interactions among the Gene Products in Support of Na+ and Alkali but Not Cholate Resistance

2000 ◽  
Vol 182 (20) ◽  
pp. 5663-5670 ◽  
Author(s):  
Masahiro Ito ◽  
Arthur A. Guffanti ◽  
Wei Wang ◽  
Terry A. Krulwich
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Vesicles ◽  
Alkali Resistance ◽  
Gene Products ◽  
Wild Type ◽  
Upstream Gene ◽  
Complex Interactions ◽  
Low Levels ◽  
The Individual ◽  
Secondary Membrane

ABSTRACT The Bacillus subtilis mrp (multiple resistance and pH) operon supports Na+ and alkali resistance via an Na+/H+ antiport, as well as cholate efflux and resistance. Among the individual mutants with nonpolar mutations in each of the seven mrp genes, only the mrpFmutant exhibited cholate sensitivity and a cholate efflux defect that were complemented by expression of the deleted gene in trans. Expression of mrpF in the mrp null (VKN1) strain also restored cholate transport and increased Na+ efflux, indicating that MrpF does not require even low levels of other mrp gene expression for its own function. In contrast to MrpF, MrpA function had earlier seemed to depend upon at least modest expression of other mrp genes, i.e., mrpA restored Na+ resistance and efflux to strain VK6 (a polar mrpA mutant which expresses low levels of mrpB to -G) but not to the null strain VKN1. In a wild-type background, each nonpolar mutation in individual mrp genes caused profound Na+sensitivity at both pH 7.0 and 8.3. The mrpA andmrpD mutants were particularly sensitive to alkaline pH even without added Na+. While transport assays in membrane vesicles from selected strains indicated that MrpA-dependent antiport can occur by a secondary, proton motive force-dependent mechanism, the requirement for multiple mrp gene products suggests that there are features of energization, function, or stabilization that differ from typical secondary membrane transporters. Northern analyses indicated regulatory relationships among mrp genes as well. All the mrp mutants, especially the mrpA,-B, -D, -E, and -G mutants, had elevated levels of mrp RNA relative to the wild type. Expression of an upstream gene, maeN, that encodes an Na+/malate symporter, was coordinately regulated withmrp, although it is not part of the operon.

scholarly journals Uncovering New Metabolic Capabilities of Bacillus subtilis Using Phenotype Profiling of Rifampin-Resistant rpoB Mutants

2007 ◽  
Vol 190 (3) ◽  
pp. 807-814 ◽  
Author(s):  
Amy E. Perkins ◽  
Wayne L. Nicholson
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Metabolic Profiling ◽  
Transcriptional Control ◽  
Β Subunit ◽  
Subunit Gene ◽  
Wild Type ◽  
Mutant Strains ◽  
Utilization Patterns ◽  
Flow Of Information

ABSTRACT RNA polymerase is a central macromolecular machine controlling the flow of information from genotype to phenotype, and insights into global transcriptional regulation can be gained by studying mutational perturbations in the enzyme. Mutations in the RNA polymerase β subunit gene rpoB causing resistance to rifampin (Rifr) in Bacillus subtilis were previously shown to lead to alterations in the expression of a number of global phenotypes known to be under transcriptional control, such as growth, competence for transformation, sporulation, and germination (H. Maughan, B. Galeano, and W. L. Nicholson, J. Bacteriol. 186:2481-2486, 2004). To better understand the global effects of rpoB mutations on metabolism, wild-type and 11 distinct congenic Rifr mutant strains of B. subtilis were tested for utilization of 95 substrates by use of Biolog GP2 MicroPlates. A number of alterations of substrate utilization patterns were observed in the Rifr mutants, including the utilization of novel substrates previously unknown in B. subtilis, such as gentiobiose, β-methyl-d-glucoside, and d-psicose. The results indicate that combining global metabolic profiling with mutations in RNA polymerase provides a system-wide approach for uncovering previously unknown metabolic capabilities and further understanding global transcriptional control circuitry in B. subtilis.

scholarly journals Biological role of the general control of amino acid biosynthesis in Saccharomyces cerevisiae.

1981 ◽  
Vol 1 (7) ◽  
pp. 584-593 ◽  
Author(s):  
P Niederberger ◽  
G Miozzari ◽  
R Hütter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Amino Acid Biosynthesis ◽  
Wild Type ◽  
General Control ◽  
Acid Biosynthesis ◽  
Mutant Strains ◽  
In The Wild ◽  
Amino Acid Imbalance

The biological role of the "general control of amino acid biosynthesis" has been investigated by analyzing growth and enzyme levels in wild-type, bradytrophic, and nonderepressing mutant strains of Saccharomyces cerevisiae. Amino acid limitation was achieved by using either bradytrophic mutations or external amino acid imbalance. In the wild-type strain noncoordinate derepression of enzymes subject to the general control has been found. Derepressing factors were in the order of 2 to 4 in bradytrophic mutant strains grown under limiting conditions and only in the order of 1.5 to 2 under the influence of external amino acid imbalance. Nonderepressing mutations led to slower growth rates under conditions of amino acid limitation, and no derepression of enzymes under the general control was observed. The amino acid pools were found to be very similar in the wild type and in nonderepressing mutant strains under all conditions tested. Our results indicate that the general control affects all branched amino acid biosynthetic pathways, namely, those of the aromatic amino acids and the aspartate family, the pathways for the basic amino acids lysine, histidine, and arginine, and also the pathways of serine and valine biosyntheses.

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