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 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 Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Matthew R. Olm ◽  
Alexander Crits-Christoph ◽  
Spencer Diamond ◽  
Adi Lavy ◽  
Paula B. Matheus Carnevali ◽  
...  
Keyword(s):  
Bacterial Diversity ◽  
Ribosomal Proteins ◽  
Large Scale ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Species Discrimination ◽  
Bacterial Genomes ◽  
Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

scholarly journals Behavior of a Wild-Type and Two Mutant Strains of Colletotrichum gloeosporioides f. sp. aeschynomene on Northern Jointvetch in the Field

Plant Disease ◽  
1998 ◽  
Vol 82 (4) ◽  
pp. 374-379 ◽  
Author(s):  
Y. Luo ◽  
D. O. TeBeest
Keyword(s):  
Population Density ◽  
Wild Type Strain ◽  
Colletotrichum Gloeosporioides ◽  
Biological Control Agent ◽  
Field Tests ◽  
The United States ◽  
Control Agent ◽  
Sampling Time ◽  
Wild Type ◽  
Mutant Strains

The fungus Colletotrichum gloeosporioides f. sp. aeschynomene causes an anthracnose on Aeschynomene virginica and has been used as a biological control agent to control this weed in the United States. The population dynamics of a wild-type strain (3-1-3) and two mutant strains of 3-1-3 of C. gloeosporioides f. sp. aeschynomene, a benomyl-resistant strain (B21) and nitrate-nonutilizing strain (Nit A), were studied in field tests on northern jointvetch in 1994 and 1995 to determine how the strains interacted on infected plants under field conditions. Plants were co-inoculated with strains 3-1-3 and B21, strains 3-1-3 and Nit A, and strains 3-1-3, B21, and Nit A at equal and unequal initial proportions. Plants were grown and maintained under flooded conditions in small wading pools. In co-inoculation of plants with 3-1-3 and B21 from equal initial proportions, the population of 3-1-3 increased slightly until it reached a proportion of 60 to 70%, whereas the population density of B21 reached 30 to 40% at the end of growing season. From unequal initial proportions, the population density of B21 decreased from 90 to about 50%, whereas the 3-1-3 increased from 10 to 50%. The population density of 3-1-3 increased from an equal initial proportion and was significantly greater than that of Nit A on every sampling time. From unequal initial proportions, the population density of 3-1-3 increased from 10 to 90%, whereas that of Nit A declined. In co-inoculation of plants with the three strains, the population density of 3-1-3 was significantly greater than those of the mutant strains at every sampling time. The proportions of mutant strains within the total population of C. gloeosporioides f. sp. aeschynomene on plants varied according to the test conditions and the number and types of strains co-inoculated.

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 Cloning, Sequencing, and Characterization of the SdeAB Multidrug Efflux Pump of Serratia marcescens

2005 ◽  
Vol 49 (4) ◽  
pp. 1495-1501 ◽  
Author(s):  
Ayush Kumar ◽  
Elizabeth A. Worobec
Keyword(s):  
Serratia Marcescens ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Library ◽  
Efflux Pump ◽  
Sodium Dodecyl ◽  
Wild Type ◽  
Diverse Range ◽  
Mutant Strains ◽  
Encoding Genes

ABSTRACT Serratia marcescens is an important nosocomial agent known for causing various infections in immunocompromised individuals. Resistance of this organism to a broad spectrum of antibiotics makes the treatment of infections very difficult. This study was undertaken to identify multidrug resistance efflux pumps in S. marcescens. Three mutant strains of S. marcescens were isolated in vitro by the serial passaging of a wild-type strain in culture medium supplemented with ciprofloxacin, norfloxacin, or ofloxacin. Fluoroquinolone accumulation assays were performed to detect the presence of a proton gradient-dependent efflux mechanism. Two of the mutant strains were found to be effluxing norfloxacin, ciprofloxacin, and ofloxacin, while the third was found to efflux only ofloxacin. A genomic library of S. marcescens wild-type strain UOC-67 was constructed and screened for RND pump-encoding genes by using DNA probes for two putative RND pump-encoding genes. Two different loci were identified: sdeAB, encoding an MFP and an RND pump, and sdeCDE, encoding an MFP and two different RND pumps. Northern blot analysis revealed overexpression of sdeB in two mutant strains effluxing fluoroquinolones. Analysis of the sdeAB and sdeCDE loci in Escherichia coli strain AG102MB, deficient in the RND pump (AcrB), revealed that gene products of sdeAB are responsible for the efflux of a diverse range of substrates that includes ciprofloxacin, norfloxacin, ofloxacin, chloramphenicol, sodium dodecyl sulfate, ethidium bromide, and n-hexane, while those of sdeCDE did not result in any change in susceptibilities to any of these agents.

scholarly journals Mutagenesis development of actinoplanes sp. KCTC 9161 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and screening for acarbose production

2018 ◽  
Vol 14 (4) ◽  
pp. 753-760
Author(s):  
Do Thi Tuyen ◽  
Nguyen The Duong ◽  
Le Thanh Hoang
Keyword(s):  
Type Ii Diabetes ◽  
Wild Type Strain ◽  
Fermentation Medium ◽  
Original Strain ◽  
Wild Type ◽  
Chromatography Method ◽  
Mutant Strains ◽  
Insulin Dependent ◽  
Mutant Lines ◽  
Layer Chromatography

Acarbose has been widely used in the therapy of type II diabetes (non-insulin dependent) because it controls blood sugar contents of patients after meals. Acarbose, a pseudo-oligosaccharide, acts as a competitive -glucosidase inhibitor. Acarbose is produced by the strains of Bacillus, Streptomyces and Actinoplanes sp. The aim of this study was to develop mutagenesis for an Actinoplanes sp. strain and screening for acarbose production. The spores of Actinoplanes sp. KCTC 9161 strain were subjected to be mutated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) for screening and finding mutant strains that were capable of production of higher acarbose (an inhibitor of α-glucosidase) higher than wild type strain. Firstly, the original NTG solution was prepared in phosphate buffer 0.05 M, pH 6.9 and the safety concentration of NTG was determined at 5 mg/ml. Then, the spores were incubated with different NTG amounts and duration. The living colonies were transferred to fermentation medium. The results obtained showed that 15 mutant strains were produced higher acarbose than wild type when used thin layer chromatography method for analysis and comparing with standard acarbose (Sigma). Three cell lines among total tested 15 mutant lines of Actinoplanes sp. KCTC 9161 produced acarbose at a higher level or indicated a higher inhibitory activity toward α-glucosidase than the original strain. Enzymatic inhibitory ativity of α-glucosidase of three mutant strains (Actinoplanes sp. KCTC- L4, L11, L14) was increased 1.3 fold higher than wild type and Actinoplanes sp. KCTC spores were very sensitive to NTG toxic, 98% spores could not survive at the treatment condition of 50 µg NTG for 30 minutes. In addition, an applicable protocol for mutating Actinoplanes sp. using NTG was suggested for further research.

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 Chemical or genetic alteration of proton motive force results in loss of virulence of Burkholderia glumae , the cause of rice bacterial panicle blight.

2021 ◽  
Author(s):  
Asif Iqbal ◽  
Pradip R. Panta ◽  
John Ontoy ◽  
Jobelle Bruno ◽  
Jong Hyun Ham ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Membrane Transporters ◽  
Motive Force ◽  
Wild Type ◽  
Bacterial Genomes ◽  
Burkholderia Glumae ◽  
Sheath Rot ◽  
Panicle Blight

Rice is an important source of food for more than half the world’s population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . B. glumae synthesizes toxoflavin, an essential virulence factor, that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis , named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae Δ dbcA strain. In addition to polymyxin sensitivity, B. glumae Δ dbcA is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild type levels). Changes in membrane potential in B. glumae Δ dbcA were reproduced in the wild type strain by the addition of sub-inhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

A mutation in an exbD gene reduces tagetitoxin production by Pseudomonas syringae pv. tagetis

2006 ◽  
Vol 52 (11) ◽  
pp. 1027-1035 ◽  
Author(s):  
Hyesuk Kong ◽  
Cheryl D Patterson ◽  
Robin E Mitchell ◽  
Jeffrey S Buyer ◽  
M Catherine Aime ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Wild Type Strain ◽  
Efflux Pump ◽  
Wild Type ◽  
Chain Reaction ◽  
Sequence Identity ◽  
Mutant Strains ◽  
Tonb System ◽  
Polymerase Chain ◽  
High Degree

A mutant of Pseudomonas syringae pv. tagetis EB037 with limited ability to produce tagetitoxin was isolated after transposon mutagenesis and the mutation was characterized. The mutation occurred in a gene with a high degree of sequence identity to exbD. exbD is contiguous with tonB and exbB upstream and with a gene for a TonB-dependent receptor downstream. Using reverse transcription – polymerase chain reaction with RNA from the wild-type and exbD mutant strains, we demonstrated that the mutation in exbD did not have a polar affect on the expression of downstream genes. The exbD mutant was able to grow well in conditions where iron is not freely available. Siderophore production by the exbD mutant was similar to that of the wild-type strain. We conclude that the mutation in exbD disrupts tagetitoxin production without compromising iron metabolism. The results indicate that tagetitoxin export by P. syringae pv. tagetis involves an efflux pump that requires a functional TonB system that is not essential for normal iron metabolism.Key words: Pseudomonas syringae pv. tagetis, Pseudomonas putida, tagetitoxin, exbD, exbB, tonB, TonB system, Helianthus annuus L.

NEW LOCI IN DICTYOSTELIUM DISCOIDEUM DETERMINING PIGMENT FORMATION AND GROWTH ON BACILLUS SUBTILIS

Genetics ◽  
1980 ◽  
Vol 96 (1) ◽  
pp. 115-123
Author(s):  
James H Morrissey ◽  
Steven Wheeler ◽  
William F Loomis
Keyword(s):  
Bacillus Subtilis ◽  
Dictyostelium Discoideum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Wild Type ◽  
Linkage Groups ◽  
New Mutation ◽  
Pigment Formation ◽  
Complementation Groups ◽  
First Time

ABSTRACT Seventeen independently isolated pigmentless (white) mutations in Dictyostelium discoideum are all recessive and fall into three complementation groups identifying two new whi loci in addition to the previously characterized whiA locus. whiB and whiC map to linkage groups III and IV, respectively. In addition, it was discovered that our laboratory stock of NC4, the wild-type strain from which these mutants were derived, has spontaneously lost the ability to grow on Bacillus subtilis. This new mutation, bsgB500, maps to linkage group VII and is not allelic to bsgA. bsgB500 is the first spontaneously derived mutation in D. discoideum that can be used to select heterozygous diploids, and for the first time allows genetic analysis to be routinely performed on strains derived from an unmutagenized background.

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