Lb464 transcripts DE in degassed and gassed beer relative to mMRS medium

Several Lb464 transcripts are SDE in both degassed and gassed beer relative to mMRS medium, that were previously implicated as important for growth in beer in a transcriptomic study of Pediococcus claussenii ATCC BAA-344 (Pc344) (37) (Table S6.1a,-b). Specifically, agmatine deiminase and putrescine carbamoyltransferase (L747_12850 and L747_12860; energy production and pH regulation), ATPase (L747_06760 and L747_07730; maintenance of proton motive force), manganese transport protein (L747_13605; hop-tolerance and oxidative stress), methionine sulfoxide reductases MsrA (L747_05475; oxidative stress), glutathione reductase (L747_11980; oxidative stress), as well as other metal transport (L747_09900) and energy homeostasis proteins (L747_06040, L747_10025, L747_05335) all are among the most highly Lb464 SDE genes in both degassed and gassed beer, as they were for Pc344 in (37). Agmantine and putrescine are among the most prevlant biogenic amines found in beer (8, 24), with the levels being affected by raw materials, brewing techniques and microbial contamination during brewing (8, 24). The formation of agmatine specifically is seen in mashing and wort boiling, and other biogenic amines are produced likely as a result of potential enzyme activity in the malt and the main fermentation (24). Both fermenting yeast and potential contamination by LAB have also been shown to procude specific biogenic amines, specifically putrescine by L. brevis (52).

The metabolism of agmatine to produce ATP, CO2, putrescine and ammonia therefore appears critical for the efficient production of energy when organisms are growing in and spoiling beer.

Several transcripts up-regulated in Lb464 likely function as structural components of the cellular membrane (“membrane proteins”), in addition to multiple general membrane transport mechanisms (ATP-binding cassette type (ABC), major facilitator superfamily (MFS) transporters, multidrug transporters, efflux (ion) pumps, and permeases), similar to what was found for Pc344 (37). Additionally, the MFS transporter horC involved in hop-tolerance (L747_00215) is up-regulated in both beer conditions as are several other transcripts found on pLb464-2. This the only Lb464 plasmid which has SDE genes above 2 Log2 FC in both beer conditions, apart from a hypothetical protein originating from pLb464-4 (L747_00880). This finding corroborates previous analyses that demonstrated pLb464-2 strongly contributed to both the hop-tolerance and beer-spoilage ability of Lb464 (12). The apparent similarities between the present Lb464 data sets and that for Pc344 (37) is important given that the study of Pc344 involved the use of

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different control medium (MRS pH 6.5 with added Tween 80, not mMRS pH 5.5), and that the dCO2 content of the beer used in the Pc344 study was not well defined (37). Thus, these commonalities lend credence to these transcripts being critical for BSR LAB survival (i.e., non-species specific) in beer.

Despite these similarities, there are several notable differences or unique processes up-regulated in Lb464 relative to Pc344 when grown in beer. For instance, the Lb464 data set reveals that there is a signal peptidase I (L747_09825) involved with processing and maturation of secretory and membrane proteins that is up regulated, whereas a similar peptidase was down-regulated in the case of Pc344. Given that several transcripts encoding Lb464 membrane proteins are shown to be SDE, it would make sense that processes involved in membrane protein maturation and functioning also are SDE. Further, the universal stress protein UspA (L747_10150) is up-regulated in Lb464 in both degassed (2.6 Log2 FC) and gassed beer (2.3 Log2 FC), whereas no specific stress response proteins were strongly expressed by Pc344 when growing in beer suggesting that Lb464 mounts a strong general stress response to the beer environment, in addition to deploying more beer-specific mechanisms.

There are also notable differences in the carbohydrate utilization and metabolism genes up-regulated in beer between the Lb464 and Pc344. For example, Lb464 expresses at greater than 2 Log2 FC key genes of operons involved with maltose metabolism (L747_07715 and L747_09905; maltose O-acetyltransferase), histidine metabolism (L747_01385;

imidazolonepropionase), arabinose metabolism (L747_07720) and butanoate metabolism (L747_08660; alpha-acetolactate decarboxylase). These processes were not implicated in the transcriptional study of Pc344 during growth in beer (37).

The up-regulation of several histidine metabolism-related genes suggests that Lb464 scavenges trace amounts of this amino acid from the environment, as it does not have direct tRNA synthesis capacity for it. Histidine can be used as a source of carbon, energy and nitrogen, and histidine metabolisms shares biosynthetic pathways with purine metabolism (i.e., L747_08270) and alanine (i.e., L747_05595), aspartate (i.e., L747_07260) and glutamate metabolism (i.e., L747_10940, L747_01690) (9), which are all processes that have transport and metabolism

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genes up-regulated in both beer media (Table S6.1-c). A recent study of beer-spoilage L. brevis isolates suggested that a specific signal transduction histidine kinase was enriched in beer-spoilage related strains, and the presence/absence of this gene was used as the first node in a dichotomic decision tree for determining beer-spoilage ability of an unknown L. brevis isolate (7). Lb464 contains one signal transduction kinase (L747_02770), which was not the specific transcripts discovered in (7), but nonetheless was SDE at 1 and 0.5 Log2 FC in degassed and gassed beer media, respectively. In previous transcriptomic data of Pc344, no significant expression of histidine kinases in beer was observed (37). Signal transduction kinases are involved in signal transduction across cellular membranes (32), and thus transcriptional data suggests some importance for a signal transduction histidine kinase during growth in unpressurized and pressurized (packaged beer) for L. brevis, however the usefulness of the specific gene suggested in (7) as a chromosomal (i.e., stable) genetic marker for L. brevis beer-spoilage ability is still unclear.

Lb464 genes involved in arabinose metabolism are up-regulated as well and given that trace amounts of arabinose have been found in beer (37), provides further testament to the nutrient scavenging ability of this bacterium. Alpha-acetolactate decarboxylase (L747_08660) is an enzyme involved with butanoate metabolism using pyruvate as a precursor, leading to the production of diacetyl and acetoin, both of which produce a buttery off-flavour in spoiled beer (50). Acetoin is also an external energy store for some fermentative bacteria, and during exponential growth can prevent the over-acidification of the cytoplasm and surrounding growth medium as a result of acidic metabolism product accumulation (i.e., acetic acid). Upon entering into the stationary phase, acetoin can be used to maintain the culture density (50). None of the carbohydrate utilization genes predicted as unique to Lb464 by RAST (Section 6.4.2.1) are shown to be SDE in beer or are up-regulated to a small extent in mMRS (L747_09170; citrate lyase and L747_10635; fructonate transporter). This is interesting given that citrate lyase metabolism is important for the biosynthesis of fatty acid during Pc344 growth in beer.

Nonetheless, Lb464 still shows SDE of pyruvate metabolism transcripts and genes involved in fatty acid production in Lb464 (i.e., L747_06040-06060, L747_11960) (Table S6.1a,-b,-c). This indicates that citrate was present in low quantities for scavenging during mid-exponential Lb464 growth and that Lb464 made use of other carbon sources to produce pyruvate and feed fatty acid

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metabolism. Again, this difference is noted with the caveat that the study of Pc344 used the same beer however with slightly different experimental conditions, however, it also indicates that the response of a given BSR LAB is likely to be individualistic based on the background genome and transcriptional regulation abilities. This finding also enforces the need for transcriptional studies to be done in parallel with comparative genomics, with both analyses done on a variety of BSR LAB with beer-spoilage ability.

One of the most distinct differences in the transcriptional pattern of Lb464 versus Pc344 when growing in beer involves the greater number of peptide and amino acid transport transcripts found to be up-regulated in Lb464 (Table S6.1a,-b,-c; Fig. 6.4). Peptide and amino acid transport has been proven advantageous to bacterial cells in defense against osmotic stress conditions (17, 51), specifically proline, which can function as water-sequestering compounds as well as chaperones for protein folding, preventing aggregation (51). Proline cannot be used by primary fermenting yeasts and has been shown to be present in finished beer, often contributing to haze-production (38). In addition, peptide and amino acid compounds and their uptake are important for nitrogen metabolism, a process known to be critical for malolactic fermentation by wine-fermenting LAB. Studies of assimilation of free available nitrogen in beer are largely concerned with the ability of fermenting yeast to utilize these compounds; in contrast, the importance of nitrogen uptake and cycling for BSR LAB is not well understood. However, the present transcriptomic data strongly indicates that nitrogen uptake and utilization is an efficient way for Lb464 to obtain energy in face of carbohydrate starvation, facilitating rapid growth (i.e., cell metabolism and replication) in beer. In addition to the increased number of peptide transport transcripts, Lb464 has several genes involved in nutrient cycling (L747_08270; xanthine permease/purine transport, L747_04210; glutamine synthetase/nitrogen metabolism, L747_07260; asparagine synthase, L747_07255; ammonia permease/transport; L747_01930l, and aminopeptidase C/amino acid processing, L747-01500 serine protease/peptide bond cleavage). There are no apparent extracellular peptidases annotated or up-regulated in beer to suggest Lb464 actively breaks down extracellular peptides or uses amino acids for scavenging.

However, there is a phosphohydrolase (L747_10665; periplasmic or membrane bound), which may facilitate breakdown of nitrogen sources in the beer environment prior to uptake into the cell.

Pc344 contains coding capacity for amino acid and peptide transport and metabolism, however

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its limited dependence on these transcripts relative to Lb464 could likely be a function of differences in genome size and coding capacity and suggests that efficient or increased nitrogen metabolism is advantageous for growth in beer.