Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1

Abstract

Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2¾10"1 and a typical transfer frequency of the order of 1¾10 "3 transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kc origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.

A Bioinformatic Study of the Distribution of Mn Oxidation Proteins in Sequenced Bacterial Genomes

“…Pseudomonas putida GB-1 has been shown to encode three separate Mn oxidase genes in its genome (21,22) and the genome of the MnOB L. discophora sp. SS-1 encodes multiple putative Mn oxidases (23). This raises the possibility that in some species Mn oxidation requires multiple enzymes working together.…”

“…The Leptothrix discophora sp. SS-1 genome encodes multiple potential Mn oxidase enzymes, including two mofA genes, two AHP genes similar to MopA, two genes predicted to encode an MCO similar to McoA and two homologs to BaMnxG (23). It is not clear how many of these proteins are capable of oxidizing Mn +2 .…”

“…It is not clear how many of these proteins are capable of oxidizing Mn +2 . Biochemical purification of the oxidase activity identified MofA as the Mn oxidase (39); however, deletion of this gene did not result in a loss of Mn oxidation (23). This raises the possibility that MofA is not a Mn oxidase; alternatively, it may be necessary to delete multiple Mn oxidation genes to lose oxidation activity, as has been observed in P. putida GB-1 (21,22).…”

“…It is not known how common it is for a bacterium to encode multiple Mn oxidase enzymes nor is it known what purpose it serves to have multiple oxidase enzymes. However, the genome of the MnOB Leptothrix discophora sp SS-1 is also predicted to encode multiple Mn oxidases (23).…”

Leptothrix cholodnii Response to Nutrient Limitation

“…To obtain ASEM snapshots without changing the medium, we also incubated SP-6 cells in a modified MSVP medium containing one-tenth the amount of sodium pyruvate (referred to as MSVP-lowC) that was supplied to maintain minimal filamentous growth. For reference, 2×YPG medium ( Bocioaga et al, 2014 ) treated or untreated with EGTA were similarly used to examine whether the filament breaks induced by the EGTA treatment were medium-dependent or not.…”