Identification of Potential Citrate Metabolism Pathways in Carnobacterium maltaromaticum

Abstract

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.

Distribution and ecology of the generalist lactic acid bacterium Carnobacterium maltaromaticum in different freshwater habitats: Metabolic and antagonistic abilities

“…A genomic characterisation based on Prokka and RAST annotations revealed that all C. maltaromaticum isolates from freshwater habitats, as well as the five published C. maltaromaticum genomes from isolates from other sources, contained genes to metabolise citrate through Pathway 2 with isocitrate and α‐ketoglutarate as intermediates (Figure 3), involving citB encoding aconitate hydratases (EC 4.2.1.3), icd encoding isocitrate dehydrogenase (EC 1.1.1.42) and citZ encoding citrate synthase (EC 2.3.3.1). Seven freshwater isolates from habitat II contained genes for citrate Pathway 1 metabolism with oxaloacetate as an intermediate, involving citF , citE and citD encoding citrate lyase alpha, beta and gamma chain (EC 4.1.3.6), citC encoding citrate (pro‐3S‐lyase) ligase (EC 6.2.1.22) and citN encoding citrate/H+ symporter of CitMHS family, in agreement with previous results (Li et al, 2021). In addition, genes for arginine degradation by the deiminase pathway, involving arcABC encoding arginine deiminase (EC 3.5.3.6), ornithine carbamoyltransferase (EC 2.1.3.3) and carbamate kinase (EC 2.7.2.2) (Arena et al, 2002) were found in all genomes as was also the case for the gene tyrDC encoding tyrosine decarboxylase (EC 4.1.1.25), catalysing the conversion of tyrosine into tyramine and carbon dioxide (Coton et al, 2004).…”

“…C. maltaromaticum also possesses other metabolic potentials, such as the utilisation of citrate. However, there is variation in whether they possess one or two potential pathways for the metabolism of this compound (Li et al, 2021). Furthermore, they are generally able to degrade arginine by the deiminase pathway and tyrosine by decarboxylation (Figure 3), both energy‐generating metabolic processes (Leisner et al, 1994; Lucas et al, 2003; Pols et al, 2021).…”

“…The phylogenetic analysis of these genes was performed using the neighbour‐joining (NJ) method by MEGA 5.0 (Kumar et al, 2008). Genes encoding H 2 O 2 formation and degradation enzymes (Andrés et al, 2022; Liu et al, 2014; Naraki et al, 2020; Streitenberger et al, 2001; Taniai et al, 2008), citrate metabolism pathway 1 and 2 (Li et al, 2021) and catabolism of arginine, tyrosine and chitin (Arena et al, 2002; Coton et al, 2004; Leisner et al, 2008) were manually searched using Prokka and RAST annotation. Genes encoding tyrosine decarboxylase not found in the automatic annotation results were searched by blasting in RAST using C. divergens 508 tyrDC sequence as a probe (Coton et al, 2004).…”

“…Subsequently, the trimmed reads were assembled using FLASH (Magoč & Salzberg, 2011). Preprocessing, including error modelling and clustering (Li et al, 2021), was performed to generate operational taxonomic units (OTUs) with QIIME (Caporaso et al, 2010). Taxonomy information for the OTUs was obtained by aligning their major sequences against the NCBI 16S database using BLASTN (v2.4.0).…”

“…The 28 C. maltaromaticum isolates (Table S5) were whole genome sequenced on an Illumina MiSeq using methodologies reported previously (Li et al, 2021). All raw sequence reads have been deposited at the NCBI under BioProject with accession PRJNA990564.…”