The transcriptome of Listeria monocytogenes during co-cultivation with cheese rind bacteria suggests adaptation by induction of ethanolamine and 1,2-propanediol catabolism pathway genes

The survival of Listeria (L.) monocytogenes in foods and food production environments (FPE) is dependent on several genes that increase tolerance to stressors; this includes competing with intrinsic bacteria. We aimed to uncover genes that are differentially expressed (DE) in L. monocytogenes sequence type (ST) 121 strain 6179 when co-cultured with cheese rind bacteria. L. monocytogenes was cultivated in broth or on plates with either a Psychrobacter or Brevibacterium isolate from cheese rinds. RNA was extracted from co-cultures in broth after two or 12 hours and from plates after 24 and 72 hours. Broth co-cultivations with Brevibacterium or Psychrobacter yielded up to 392 and 601 DE genes, while plate co-cultivations significantly affected the expression of up to 190 and 485 L. monocytogenes genes, respectively. Notably, the transcription of virulence genes encoding the Listeria adhesion protein and Listeriolysin O were induced during plate and broth co-cultivations. The expression of several systems under the control of the global stress gene regulator, σB, increased during co-cultivation. A cobalamin-dependent gene cluster, responsible for the catabolism of ethanolamine and 1,2-propanediol, was upregulated in both broth and plate co-cultures conditions. Finally, a small non-coding (nc)RNA, Rli47, was induced after 72 hours of co-cultivation on plates and accounted for 50–90% of the total reads mapped to L. monocytogenes. A recent study has shown that Rli47 may contribute to L. monocytogenes stress survival by slowing growth during stress conditions through the suppression of branch-chained amino acid biosynthesis. We hypothesize that Rli47 may have an impactful role in the response of L. monocytogenes to co-cultivation by regulating a complex network of metabolic and virulence mechanisms.


RNA extraction, transcriptome sequencing, and analysis
132 RNA used in transcriptome sequencing was obtained using the Invitrogen Purelink RNA Mini kit.

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After cultivation, broth tube samples were centrifuged at 4696 x g for 3 minutes at 20°C and pellets was verified using an RNA 6000 Nano chip on an Agilent 2100 Bioanalyzer (Prokaryote Total sequencing platform, and demultiplexing and trimming of Illumina adaptor sequences were the respective condition control consistently demonstrated that replicates of shorter co-179 cultivations were more variable. In contrast, replicates of longer cultivations clustered much closer 180 together by condition (i.e., co-culture replicates clustered closely together and separate from the 181 pure culture control replicates), indicating less variability within a condition and a stronger 182 treatment effect (S1 and S2 Figs). Among all experiments, the number of DE genes (Fig 1) and 183 associated log2 fold changes of co-cultivation conditions ranged from 0 to 601 and -6.96 to 8.41, 184 respectively (S1 and S2 Tables   278 monocytogenes 6179 and Psychrobacter L7, averaging 29 x and 117 x, respectively (Table 3).

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M18, a Brevibacterium linens anti-listerial bacteriocin, was expressed by Brevibacterium S111 in Brevibacterium S111 gene expression would be required to validate this hypothesis.

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Plasmid gene expression and plasmid coverage ranged from 4 to 89 x (Tables 3 and 4). L. monocytogenes 6179 exposed 386 to co-culture conditions induced the differential expression of seven plasmid genes ( 396 found that during mild acid stress, uvrX expression was highly strain-dependent.  The co-cultivation of L. monocytogenes 6179 with Psychrobacter L7 or Brevibacterium S111 428 induced differential expression patterns of several genes and pathways conserved among 429 multiple co-cultivation conditions, which are described below and summarized in S4

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The present study reveals that L. monocytogenes is altering expression of aerobic and anaerobic 520 respiratory and pyruvate fermentation pathways in response to co-culture conditions. Changing 521 the expression of various metabolic pathway genes may enable L. monocytogenes to adapt better 522 to nutrient availability during co-culture, and it seems that Rli47 may have a general role in 523 modulating these changes.

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Pyrimidine biosynthesis 525 A gene cluster (pyr genes) annotated as being involved in pyrimidine biosynthesis and salvage 526 was upregulated in both 12 h co-cultivations, 24 h plate co-cultivation with Psychrobacter L7, and 527 72 h of co-cultivation with Brevibacterium S111 (Table 6). We hypothesize that L. monocytogenes 537 monocytogenes could replace glutamine with ethanolamine as a nitrogen source. Therefore, we 538 argue that nitrogen derived from the pyrimidine biosynthesis and ethanolamine degradation gene 539 clusters may confer a selective advantage for L. monocytogenes during nitrogen starvation as a 541  Fig 2), ethanolamine (eut genes, Fig   548  3), and the import and biosynthesis of cobalamin (cbi/cob genes Fig 4). Cobalamin is an essential     643 obtained in the current study, we have developed a scenario of pathways found to be consistently 644 upregulated during coculture and where Rli47 may influence these systems (Fig 5). Further 645 studies will be necessary to elucidate how the genes and pathways of interest identified in this 646 study may contribute to L. monocytogenes fitness when exposed to other bacteria.