Genome Sequence of Megasphaera cerevisiae NSB1, a Bacterium Isolated from a Canning Line and Able To Grow in Beer with High Alcohol Content.

The genome sequence of the brewery isolate Megasphaera cerevisiae NSB1 was determined. Strain NSB1 tolerates 5% (vol/vol) alcohol, which is higher than previously reported for M. cerevisiae The NSB1 genome will help elucidate genetics required for alcohol tolerance and niche adaptation of this Gram-negative beer-spoilage bacterium.

GENOME ANNOUNCEMENT

Beer spoilage by the anaerobic Gram-negative bacterium Megasphaera cerevisiae is severe, as it produces strong off-flavors and aromas such as acetoin and butyric, acetic, caproic, isovaleric, and caproic acids (1). M. cerevisiae contamination is reportedly limited to beers with low alcohol content (<3.5% wt/vol) (2). However, M. cerevisiae NSB1, as isolated from a canning line, is able to grow in and spoil hopped ale containing 5% (vol/vol) ethanol with 45 international bitterness units and pH 4.55. M. cerevisiae NSB1 was propagated on agar plates of WL Differential media (HiMedia Laboratories, Mumbai, India), containing 4 mg/L cycloheximide and 0.5 g/L cysteine hydrochloride with anaerobic incubation at 30°C. Microbial mass was suspended in MicroBead Solution of a MoBio UltraClean DNA extraction kit (Mo Bio Laboratories, Carlsbad, CA, USA). DNA extraction proceeded according to the manufacturer’s instructions, including an optional heating step of 70°C for 10 min prior to bead beating to increase cell lysis efficiency.

Sequencing was performed by Illumina MiSeq (paired-end 2 × 300 bp; at Contango Strategies Ltd., Saskatoon, SK, Canada). Reads were assembled by SPAdes-3.9.0-Darwin (3), using the “–careful” flag to minimize the number of mismatches. Assembly output was filtered to remove scaffolds <450 bp and coverage scores <2, producing 163 contigs with 40× coverage and an N50 length of 48,664. The draft genome sequence is 3,015,877 bp long, with a 45% G+C content. Annotation was performed by RAST (Rapid Annotations using Subsystems Technology) version 2.0 (4) via the “RASTtk” modular pipeline (5) and PGAP (NCBI Prokaryotic Genome Annotation Pipeline) (6). The PGAP annotation revealed that M. cerevisiae NSB1 has 2,827 genes and 2,652 protein-coding sequences, 19 rRNA operon (CDSs), 53 tRNAs, one CRISPR array, and 89 pseudogenes.

M. cerevisiae NSB1 contains a complete suf operon (involved in iron homeostasis) and the manganese transporter MntH, as described for the genome-sequenced beer-spoilage strain M. cerevisiae PAT 1T (DSM 20462T) (7). Unlike in PAT 1T, the full arginine catabolism operon is not complete in M. cerevisiae NSB1. Conversely, M. cerevisiae NSB1 contains several CDSs not present in PAT 1T, including a cadmium-transporting ATPase, and a complete anaerobic sulfite reductase operon (asrA, B, and C) involved in iron and sulfite homeostasis. Additionally, several transcripts related to capsular and extracellular polysaccharides metabolism are exclusive to M. cerevisiae NSB1.

M. cerevisiae NSB1 also contains numerous transcripts related to lactate utilization (l-lactate dehydrogenase and l-lactate permease), which are involved in producing acetic, butyric, and hexanoic acid in M. elsdenii (8). Glycerol kinase and glycerol dehydratase pathways, responsible for glycerol degradation, are also present in M. cerevisiae NSB1, likely further contributing to beer off-flavor through production of highly volatile acid and aroma compounds, as has been found for the Gram-positive beer-spoilage organism Pediococcus pentosaceus CAg (9). A large assortment of multidrug efflux pump genes are present, as are multiple copies of alcohol dehydrogenase genes, which all may contribute to the export of solvent/ethanol, thus explaining the unusual high-alcohol tolerance of M. cerevisiae NSB1. The genetics of M. cerevisiae NSB1 are worthy of further exploration from the perspective of understanding the beer-spoilage capacity of M. cerevisiae.

Accession number(s).

The M. cerevisiae NSB1 genome was deposited in GenBank under the accession number MQNM00000000.