Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG- 1

Protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation

L. G. Van Waasbergen, J. A. Hoch, Bradley Tebo

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Abstract

Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (~100 transformants per μg of DNA) with several different plasmids of gram- positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10-3. Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.

Original languageEnglish (US)
Pages (from-to)7594-7603
Number of pages10
JournalJournal of Bacteriology
Volume175
Issue number23
StatePublished - 1993
Externally publishedYes

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Protoplasts
Manganese
Mutagenesis
Bacillus
Plasmids
Spores
Genetic Phenomena
Capsid Proteins
Chromosomes
Temperature
DNA
Genes

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Immunology

Cite this

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title = "Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG- 1: Protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation",
abstract = "Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (~100 transformants per μg of DNA) with several different plasmids of gram- positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98{\%} plasmid loss and insertions to be recovered at a frequency of 10-3. Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.",
author = "{Van Waasbergen}, {L. G.} and Hoch, {J. A.} and Bradley Tebo",
year = "1993",
language = "English (US)",
volume = "175",
pages = "7594--7603",
journal = "Journal of Bacteriology",
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TY - JOUR

T1 - Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG- 1

T2 - Protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation

AU - Van Waasbergen, L. G.

AU - Hoch, J. A.

AU - Tebo, Bradley

PY - 1993

Y1 - 1993

N2 - Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (~100 transformants per μg of DNA) with several different plasmids of gram- positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10-3. Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.

AB - Mature spores of the marine Bacillus sp. strain SG-1 bind and oxidize manganese(II), thereby becoming encrusted with a manganese(IV) oxide. Both the function and mechanism of this oxidation are unknown, although evidence suggests that spore coat proteins are involved. To further study this phenomenon, methods of genetic analysis were developed for SG-1. By a modified protoplast transformation procedure, SG-1 was transformed (~100 transformants per μg of DNA) with several different plasmids of gram- positive origin. Transposon Tn917, delivered on the temperature-sensitive plasmid pLTV1, was used to generate mutants of SG-1. Conditions were established that allowed 98% plasmid loss and insertions to be recovered at a frequency of 10-3. Each mutant was found to be the result of a single insertion event. Restriction analysis of 27 mutants that do not oxidize manganese but still sporulate localized 17 of the insertions within two regions of the chromosome (termed Mnx regions), and a physical map of these regions was generated. Analysis of 18 transposon integrants in which manganese oxidation was unaffected revealed random transposon integration, with none of their insertions mapping within the Mnx regions. The Mnx regions were cloned from wild-type SG-1, and the largest region, carried on the lactococcal plasmid pGK13, was used to complement in trans one of the nonoxidizing mutants. These results demonstrate that the Mnx regions encode factors that are required for the oxidation of manganese, and this represents the first report identifying genes involved in bacterial manganese oxidation.

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