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 Bacillus subtilis mutants harbouring a single copy of the rRNA operon exhibit severe defects in growth and sporulation

Microbiology ◽  
2010 ◽  
Vol 156 (10) ◽  
pp. 2944-2952 ◽  
Author(s):  
Hideaki Nanamiya ◽  
Makiko Sato ◽  
Kenta Masuda ◽  
Mikiko Sato ◽  
Tetsuya Wada ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Functional Significance ◽  
Wild Type Strain ◽  
Bacterial Species ◽  
Single Copy ◽  
Rrna Operon ◽  
Rrna Genes ◽  
Wild Type ◽  
Bacterial Genomes ◽  
Mutant Strains

The number of copies of rRNA genes in bacterial genomes differs greatly among bacterial species. It is difficult to determine the functional significance of the heterogeneity of each rRNA operon fully due to the existence of multiple rRNA operons and because the sequence heterogeneity among the rRNA genes is extremely low. To overcome this problem, we sequentially deleted the ten rrn operons of Bacillus subtilis and constructed seven mutant strains that each harboured a single rrn operon (either rrnA, B, D, E, I, J or O) in their genome. The growth rates and sporulation frequencies of these mutants were reduced drastically compared with those of the wild-type strain, and this was probably due to decreased levels of ribosomes in the mutants. Interestingly, the ability to sporulate varied significantly among the mutant strains. These mutants have proved to be invaluable in our initial attempts to reveal the functional significance of the heterogeneity of each rRNA operon.

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 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 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 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 Regulation of the Bacillus subtilis Extracytoplasmic Function Protein σY and Its Target Promoters

2003 ◽  
Vol 185 (16) ◽  
pp. 4883-4890 ◽  
Author(s):  
Min Cao ◽  
Letal Salzberg ◽  
Ching Sung Tsai ◽  
Thorsten Mascher ◽  
Carla Bonilla ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Sigma Factor ◽  
Negative Regulation ◽  
Wild Type ◽  
Induced Mutations ◽  
Mutant Strains ◽  
Elevated Expression ◽  
Direct Target ◽  
Extracytoplasmic Function Sigma Factor ◽  
Target Promoters

ABSTRACT The Bacillus subtilis extracytoplasmic function sigma factor σY is of unknown function. We demonstrate that the sigY operon is expressed from an autoregulatory promoter site, PY. We selected for transposon-induced mutations that upregulate PY transcription in an attempt to identify genes involved in σY regulation. The resulting insertions disrupted yxlC, the gene immediately downstream of sigY. However, the phenotype of the yxlC::Tn10 insertion was due to polarity on the downstream genes of the sigY operon; a nonpolar insertion in yxlC did not lead to derepression of PY. Further analyses revealed that both yxlD and yxlE encoded proteins important for the negative regulation of σY activity. A comparison of the transcriptomes of wild-type and yxlC::Tn10 mutant strains revealed elevated expression of several operons. However, only one additional gene, ybgB, was unambiguously identified as a direct target for σY. This was supported by analysis of direct targets for σY transcription with whole-genome runoff transcription followed by macroarray analysis.

scholarly journals Role of anionic phospholipids in the adaptation of Bacillus subtilis to high salinity

Microbiology ◽  
2006 ◽  
Vol 152 (3) ◽  
pp. 605-616 ◽  
Author(s):  
Claudia S. López ◽  
Alejandro F. Alice ◽  
Horacio Heras ◽  
Emilio A. Rivas ◽  
Carmen Sánchez-Rivas
Keyword(s):  
Bacillus Subtilis ◽  
Stationary Phase ◽  
Growth Curves ◽  
Membrane Lipid ◽  
Wild Type ◽  
Lipid Packing ◽  
Anionic Phospholipids ◽  
Large Unilamellar Vesicles ◽  
Mutant Strains ◽  
Lag Period

The importance of the content of anionic phospholipids [cardiolipin (CL) and phosphatidylglycerol (PG)] in the osmotic adaptation and in the membrane structure of Bacillus subtilis cultures was investigated. Insertion mutations in the three putative cardiolipin synthase genes (ywiE, ywnE and ywjE) were obtained. Only the ywnE mutation resulted in a complete deficiency in cardiolipin and thus corresponds to a true clsA gene. The osmotolerance of a clsA mutant was impaired: although at NaCl concentrations lower than 1·2 M the growth curves were similar to those of its wild-type control, at 1·5 M NaCl (LBN medium) the lag period increased and the maximal optical density reached was lower. The membrane of the clsA mutant strain showed an increased PG content, at both exponential and stationary phase, but no trace of CL in either LB or LBN medium. As well as the deficiency in CL synthesis, the clsA mutant showed other differences in lipid and fatty acids content compared to the wild-type, suggesting a cross-regulation in membrane lipid pathways, crucial for the maintenance of membrane functionality and integrity. The biophysical characteristics of membranes and large unilamellar vesicles from the wild-type and clsA mutant strains were studied by Laurdan's steady-state fluorescence spectroscopy. At physiological temperature, the clsA mutant showed a decreased lateral lipid packing in the protein-free vesicles and isolated membranes compared with the wild-type strain. Interestingly, the lateral lipid packing of the membranes of both the wild-type and clsA mutant strains increased when they were grown in LBN. In a conditional IPTG-controlled pgsA mutant, unable to synthesize PG and CL in the absence of IPTG, the osmoresistance of the cultures correlated with their content of anionic phospholipids. The transcriptional activity of the clsA and pgsA genes was similar and increased twofold upon entry to stationary phase or under osmotic upshift. Overall, these results support the involvement of the anionic phospholipids in the growth of B. subtilis in media containing elevated NaCl concentrations.

scholarly journals Physiological Effects of Anti-TRAP Protein Activity and tRNATrp Charging on trp Operon Expression in Bacillus subtilis

2008 ◽  
Vol 190 (6) ◽  
pp. 1937-1945 ◽  
Author(s):  
Luis R. Cruz-Vera ◽  
Ming Gong ◽  
Charles Yanofsky
Keyword(s):  
Bacillus Subtilis ◽  
Growth Rates ◽  
Transcription Termination ◽  
Physiological Responses ◽  
Physiological Effects ◽  
Deficient Mutant ◽  
Wild Type ◽  
Protein Activity ◽  
Mutant Strains ◽  
Operon Expression

ABSTRACT The Bacillus subtilis anti-TRAP protein regulates the ability of the tryptophan-activated TRAP protein to bind to trp operon leader RNA and promote transcription termination. AT synthesis is regulated both transcriptionally and translationally by uncharged tRNATrp. In this study, we examined the roles of AT synthesis and tRNATrp charging in mediating physiological responses to tryptophan starvation. Adding excess phenylalanine to wild-type cultures reduced the charged tRNATrp level from 80% to 40%; the charged level decreased further, to 25%, in an AT-deficient mutant. Adding tryptophan with phenylalanine increased the charged tRNATrp level, implying that phenylalanine, when added alone, reduces the availability of tryptophan for tRNATrp charging. Changes in the charged tRNATrp level observed during growth with added phenylalanine were associated with increased transcription of the genes of tryptophan metabolism. Nutritional shift experiments, from a medium containing tryptophan to a medium with phenylalanine and tyrosine, showed that wild-type cultures gradually reduced their charged tRNATrp level. When this shift was performed with an AT-deficient mutant, the charged tRNATrp level decreased even further. Growth rates for wild-type and mutant strains deficient in AT or TRAP or that overproduce AT were compared in various media. A lack of TRAP or overproduction of AT resulted in phenylalanine being required for growth. These findings reveal the importance of AT in maintaining a balance between the synthesis of tryptophan versus the synthesis of phenylalanine, with the level of charged tRNATrp acting as the crucial signal regulating AT production.

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.

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