Mining Biosynthetic Gene Clusters in Carnobacterium maltaromaticum by Interference Competition Network and Genome Analysis.

Carnobacterium maltaromaticum is a non-starter lactic acid bacterium (LAB) of interest in the dairy industry for biopreservation. This study investigated the interference competition network and the specialized metabolites biosynthetic gene clusters (BGCs) content in this LAB in order to explore the relationship between the antimicrobial properties and the genome content. Network analysis revealed that the potency of inhibition tended to increase when the inhibition spectrum broadened, but also that several strains exhibited a high potency and narrow spectrum of inhibition. The C. maltaromaticum strains with potent anti-L. monocytogenes were characterized by high potency and a wide intraspecific spectrum. Genome mining of 29 strains revealed the presence of 12 bacteriocin BGCs: four of class I and eight of class II, among which seven belong to class IIa and one to class IIc. Overall, eight bacteriocins and one nonribosomal peptide synthetase and polyketide synthase (NRPS-PKS) BGCs were newly described. The comparison of the antimicrobial properties resulting from the analysis of the network and the BGC genome content allowed us to delineate candidate BGCs responsible for anti-L. monocytogenes and anti-C. maltaromaticum activity. However, it also highlighted that genome analysis is not suitable in the current state of the databases for the prediction of genes involved in the antimicrobial activity of strains with a narrow anti-C. maltaromaticum activity.

Identification of Potential Citrate Metabolism Pathways in Carnobacterium maltaromaticum.

In the present study, we describe the identification of potential citrate metabolism pathways for the lactic acid bacterium (LAB) Carnobacterium maltaromaticum. A phenotypic assay indicated that four of six C. maltaromaticum strains showed weak (Cm 6-1 and ATCC 35586) or even delayed (Cm 3-1 and Cm 5-1) citrate utilization activity. The remaining two strains, Cm 4-1 and Cm 1-2 gave negative results. Additional analysis showed no or very limited utilization of citrate in media containing 1% glucose and 22 or 30 mM citrate and inoculated with Cm 6-1 or ATCC 35586. Two potential pathways of citrate metabolism were identified by bioinformatics analyses in C. maltaromaticum including either oxaloacetate (pathway 1) or tricarboxylic compounds such as isocitrate and α-ketoglutarate (pathway 2) as intermediates. Genes encoding pathway 1 were present in two out of six strains while pathway 2 included genes present in all six strains. The two potential citrate metabolism pathways in C. maltaromaticum may potentially affect the sensory profiles of milk and soft cheeses subjected to growth with this species.

Nested structure of intraspecific competition network in Carnobacterium maltaromaticum.

While competition targeting food-borne pathogens is being widely documented, few studies have focused on competition among non-pathogenic food bacteria. Carnobacterium maltaromaticum is a genetically diverse lactic acid bacterium known for comprising several bacteriocinogenic strains with bioprotective potentialities against the food-borne pathogen Listeria monocytogenes. The aim of our study is to examine the network properties of competition among a collection of 73 strains of C. maltaromaticum and to characterize their individual interaction potential. The performed high-throughput competition assays, investigating 5 329 pairwise interactions, showed that intraspecific competition was major in C. maltaromaticum with approximately 56% of the sender strains antagonizing at least one receiver strain. A high diversity of inhibitory and sensitivity spectra was identified along with a majority of narrow inhibitory as well as sensitivity spectra. Through network analysis approach, we determined the highly nested architecture of C. maltaromaticum competition network, thus showing that competition in this species is determined by both the spectrum width of the inhibitory activity of sender strains and the spectrum width of the sensitivity of receiver strains. This study provides knowledge of the competition network in C. maltaromaticum that could be used in rational assembly of compatible microbial strains for the design of mixed starter cultures.

High-Throughput Identification of Candidate Strains for Biopreservation by Using Bioluminescent Listeria monocytogenes.

This article describes a method for high-throughput competition assays using a bioluminescent strain of L. monocytogenes. This method is based on the use of the luminescent indicator strain L. monocytogenes EGDelux. The luminescence of this strain is correlated to growth, which make it suitable to monitor the growth of L. monocytogenes in mixed cultures. To this aim, luminescence kinetics were converted into a single numerical value, called the Luminescence Disturbance Indicator (LDI), which takes into account growth inhibition phenomena resulting in latency increase, decrease in the luminescence rate, or reduction of the maximum luminescence. The LDI allows to automatically and simultaneously handle multiple competition assays which are required for high-throughput screening (HTS) approaches. The method was applied to screen a collection of 1810 strains isolated from raw cow's milk in order to identify non-acidifying strains with anti-L. monocytogenes bioprotection properties. This method was also successfully used to identify anti-L. monocytogenes candidates within a collection of Lactococcus piscium, a species where antagonism was previously described as non-diffusible and requiring cell-to-cell contact. In conclusion, bioluminescent L. monocytogenes can be used in HTS to identify strains with anti-L. monocytogenes bioprotection properties, irrespectively of the inhibition mechanism.