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A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium Macrococcus caseolyticus subsp. caseolyticus.

ABSTRACT: Macrococcus caseolyticus subsp. caseolyticus is a Gram-positive, commensal organism documented to be present as a component of the secondary microflora in fermented foods such as Ragusano and Fontina cheeses and Cantonese sausage. In these products, the organism appears to play a role in ripening and the development of the final organoleptic qualities. However, the role of this organism in flavor generation is not well understood. Therefore, the objective of this study was to investigate the role of M. caseolyticus subsp. caseolyticus in flavor compound formation through an examination of enzymatic, metabolomic and genomic data. A bank of M. caseolyticus subsp. caseolyticus strains derived from a variety of niches were examined. Enzyme activities analyzed comprised those of the proteolytic and lipolytic cascades including cell-envelope proteinase (CEP), peptidases, esterases, lipases, aminotransferases and glutamate dehydrogenase (GDH). Strain to strain variation was observed, often associated with niche. All strains, except those isolated from non-dairy sources, demonstrated high CEP activity. Such high CEP activity associated with dairy strains implies the importance of this characteristic in the adaptation of these strains to a dairy-specific niche. However, limited downstream peptidolytic activity, in addition to a limited ability to generate free amino acids (FAA) was observed across all strains, indicating weak ability of this organism to generate amino-acid derived flavor compounds. Interestingly, the strains with high CEP activity also demonstrated high esterase activity and gas chromatography-mass spectrometry (GC-MS) analysis of the volatile compounds produced when these strains were grown in lactose-free milk demonstrated differences in the range and types of volatiles produced. In contrast to this metabolic versatility, comparative genome analysis revealed the distribution of components of the proteolytic and lipolytic system in these strains to be conserved. Overall, this study demonstrates the potential of M. caseolyticus subsp. caseolyticus to generate diverse volatile flavor compounds. Additionally, the identification of the highly active strain-specific cell wall bound caseolytic proteases deriving extensive casein hydrolysis, serves as a promising avenue which can be potentially harnessed in the future to produce greater and more diverse flavor compounds.

SUBMITTER: Mazhar S

PROVIDER: S-EPMC7358451 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium Macrococcus caseolyticus subsp. caseolyticus.

Mazhar Shahneela S Kilcawley Kieran N KN Hill Colin C McAuliffe Olivia O

Frontiers in microbiology 20200707

Macrococcus caseolyticus subsp. caseolyticus is a Gram-positive, commensal organism documented to be present as a component of the secondary microflora in fermented foods such as Ragusano and Fontina cheeses and Cantonese sausage. In these products, the organism appears to play a role in ripening and the development of the final organoleptic qualities. However, the role of this organism in flavor generation is not well understood. Therefore, the objective of this study was to inves ...[more]

PMID: 32733419

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Project description:The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

Project description:Macrococcus caseolyticus belongs to the normal bacterial flora of dairy cows and does not usually cause disease. However, methicillin-resistant M. caseolyticus strains were isolated from bovine mastitis milk. These bacteria had acquired a chromosomal island (McRI mecD -1 or McRI mecD -2) carrying the methicillin resistance gene mecD To gain insight into the distribution of McRI mecD types in M. caseolyticus from cattle, 33 mecD-containing strains from Switzerland were characterized using molecular techniques, including multilocus sequence typing, antibiotic resistance gene identification, and PCR-based McRI mecD typing. In addition, the same genetic features were analyzed in 27 mecD-containing M. caseolyticus strains isolated from bovine bulk milk in England/Wales using publicly available whole-genome sequences. The 60 strains belonged to 24 different sequence types (STs), with strains belonging to ST5, ST6, ST21, and ST26 observed in both Switzerland and England/Wales. McRI mecD -1 was found in different STs from Switzerland (n?=?19) and England/Wales (n?=?4). McRI mecD -2 was only found in 7 strains from Switzerland, all of which belonged to ST6. A novel island, McRI mecD -3, which contains a complete mecD operon (mecD-mecR1m-mecIm [where the subscript m indicates Macrococcus]) combined with the left part of McRI mecD -2 and the right part of McRI mecD -1, was found in heterogeneous STs from both collections (Switzerland, n?=?7; England/Wales, n?=?21). Two strains from England/Wales carried a truncated McRI mecD -3. Phylogenetic analyses revealed no clustering of strains according to geographical origin or carriage of McRI mecD -1 and McRI mecD -3. Circular excisions were also detected for McRI mecD -1 and McRI mecD -3 by PCR. The analyses indicate that these islands are mobile and may spread by horizontal gene transfer between genetically diverse M. caseolyticus strains.IMPORTANCE Since its first description in 2017, the methicillin resistance gene mecD has been detected in M. caseolyticus strains from different cattle sources and countries. Our study provides new insights into the molecular diversity of mecD-carrying M. caseolyticus strains by using two approaches to characterize mecD elements: (i) multiplex PCR for molecular typing of McRI mecD and (ii) read mapping against reference sequences to identify McRI mecD types in silico In combination with multilocus sequence typing, this approach can be used for molecular characterization and surveillance of M. caseolyticus carrying mecD.

Project description:Macrococcus caseolyticus is an opportunistic pathogen that is frequently isolated from dairy products and veterinary infections. Recent studies have reported the possibility of methicillin resistance that be transferred among staphylococcal species in foods. The present study examined the population structure, antimicrobial resistance, virulence factors, and morphology of methicillin-resistant M. caseolyticus by investigation of 94 genomes derived from both isolates in beef (n = 7) and pork (n = 2) at Shanghai and those deposited in public domain (n = 85). Phylogenetically, M. caseolyticus were divided into four clades, which each consisted of genomes isolated from continent of European countries (82.4%, n = 78), Asian countries (11.3%, n = 10), United States (4.1%, n = 4), Australia (1%, n = 1), and Sudan (1%, n = 1). The M. caseolyticus isolated from present study formed a genetically distinguished clade, which was characterized by novel alleles in the traditional 7-gene MLST scheme. Furthermore, we identified 24 AMR genes that were associated with 10 classes of antimicrobial agents in M. caseolyticus. Most AMR genes were carried by dominant plasmids such as rep7a, rep22 and repUS56. The genomes in the global clades carried significantly less AMR genes (p < 0.05) and more virulence factors (p < 0.001) than present clade. Virulence factors were detected in methicillin resistant M. caseolyticus including genes coding hemolysin, adherence, biofilm formation, exotoxin, and capsule that associated to human health and infection. Finally, as the close relative of the genus Staphylococcus, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were performed for morphological comparison that M. caseolyticus has a larger diameter and thicker cell wall compared with S. aureus ATCC 25,923. Taken together, our study suggested that M. caseolyticus mediating divergent antimicrobial resistance and virulence factors could serve as the vector for methicillin resistance habitats in foodborne microorganisms.

Dataset Information

A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium Macrococcus caseolyticus subsp. caseolyticus.

Publications

A Systems-Wide Analysis of Proteolytic and Lipolytic Pathways Uncovers The Flavor-Forming Potential of The Gram-Positive Bacterium <i>Macrococcus caseolyticus</i> subsp. <i>caseolyticus</i>.

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