Draft Genome Sequences of Two Lactobacillus casei Strains Isolated from Cheddar Cheese and a Fermented Milk Drink

Nahar, Akhikun; Baker, Anthony L.; Bowman, John P.; Britz, ML

doi:10.1128/genomea.01235-17

Cited by 4 publications

(13 citation statements)

References 10 publications

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“…casei W56 or from the RAST SEED finder annotation of Lb . casei GCRL163 genome [ 27 ], with validation by BLASTP in KEGG and NCBI ** Denotes very low LFQ intensity (log 2- transformed values <<20). …”

Section: Resultsmentioning

confidence: 99%

“…casei W56 or from the RAST SEED finder annotation of Lb . casei GCRL163 genome [ 27 ], with validation by BLASTP in KEGG and NCBI…”

Section: Resultsmentioning

confidence: 99%

“…BLASTP (KEGG) of the Lb . casei GCRL163 SecB homolog (protein sequence from the RAST file of the sequenced genome [ 27 ] showed 100% sequence similarity with Lb . casei and Lb .…”

Section: Resultsmentioning

confidence: 99%

“…casei strain W56 (ID UP000003734) database comprising 3,092 protein entries ( www.uniprot.org ) using the Andromeda search engine, since the genome of Lb . casei GCRL163 has been shotgun sequenced (DDBJ/EMBL/GenBank accession number MODT00000000) but not closed [ 27 ]. Hence, Lb .…”

Section: Methodsmentioning

confidence: 99%

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Proteomic analysis of Lactobacillus casei GCRL163 cell-free extracts reveals a SecB homolog and other biomarkers of prolonged heat stress

et al. 2018

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Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163.

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Section: Resultsmentioning

confidence: 99%

“…casei W56 or from the RAST SEED finder annotation of Lb . casei GCRL163 genome [ 27 ], with validation by BLASTP in KEGG and NCBI…”

Section: Resultsmentioning

confidence: 99%

“…BLASTP (KEGG) of the Lb . casei GCRL163 SecB homolog (protein sequence from the RAST file of the sequenced genome [ 27 ] showed 100% sequence similarity with Lb . casei and Lb .…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Proteomic analysis of Lactobacillus casei GCRL163 cell-free extracts reveals a SecB homolog and other biomarkers of prolonged heat stress

et al. 2018

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“…L. paracasei VUP 12006 was initially stored in the Victoria University, Werribee, VIC, Australia, culture collection [32] and was renamed GCRL 46 when re-stored in the University of Melbourne, Gilbert Chandler Research Laboratories, Werribee, VIC, Australia [32]. L. casei GCRL 163 was originally isolated from maturing Cheddar cheese [64] and its genome was published recently [65]. Strains were cultured routinely using de Man, Rogosa, Sharpe (MRS) broth (Oxoid, Thebarton, SA, Australia) or plates (1.5% agar, Oxoid) and incubated at 37 °C under anaerobic conditions using Oxoid jars with Gas Generating Kit BR038B.…”

Section: Methodsmentioning

confidence: 99%

An Acid Up-Regulated Surface Protein of Lactobacillus paracasei Strain GCRL 46 is Phylogenetically Related to the Secreted Glucan- (GpbB) and Immunoglobulin-Binding (SibA) Protein of Pathogenic Streptococci

Pepper

Britz

2019

IJMS

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Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40–45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

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Prolonged Heat Stress of Lactobacillus paracasei GCRL163 Improves Binding to Human Colorectal Adenocarcinoma HT-29 Cells and Modulates the Relative Abundance of Secreted and Cell Surface-Located Proteins

et al. 2020

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Lactobacillus casei group bacteria improve cheese ripening and may interact with host intestinal cells as probiotics, where surface proteins play a key role. Three complementary methods [trypsin shaving (TS), LiCl−sucrose (LS) extraction, and extracellular culture fluid precipitation] were used to analyze cell surface proteins of Lactobacillus paracasei GCRL163 by label-free quantitative proteomics after culture to the mid-exponential phase in bioreactors at pH 6.5 and temperatures of 30−45 °C. A total of 416 proteins, including 300 with transmembrane, cell wall anchoring, and secretory motifs and 116 cytoplasmic proteins, were quantified as surface proteins. Although LS caused significantly greater cell lysis as growth temperature increased, higher numbers of extracytoplasmic proteins were exclusively obtained by LS treatment. Together with the increased positive surface charge of cells cultured at supra-optimal temperatures, proteins including cell wall hydrolases Msp1/p75 and Msp2/p40, α-fucosidase AlfB, SecA, and a PspC-domain putative adhesin were upregulated in surface or secreted protein fractions, suggesting that cell adhesion may be altered. Prolonged heat stress (PHS) increased binding of L. paracasei GCRL163 to human colorectal adenocarcinoma HT-29 cells, relative to acid-stressed cells. This study demonstrates that PHS influences cell adhesion and relative abundance of proteins located on the surface, which may impact probiotic functionality, and the detected novel surface proteins likely linked to the cell cycle and envelope stress.

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Draft Genome Sequences of Two Lactobacillus casei Strains Isolated from Cheddar Cheese and a Fermented Milk Drink

Abstract: MiSeq Illumina shotgun sequencing technology was used to sequence two Lactobacillus casei strains, designated strains GCRL 163 and MJA 12. The estimated genome sizes for GCRL 163 and MJA 12 were 2.9 Mb and 3.1 Mb, with 46.35% and 46.31% GC contents, respectively.

Cited by 4 publications

References 10 publications

Proteomic analysis of Lactobacillus casei GCRL163 cell-free extracts reveals a SecB homolog and other biomarkers of prolonged heat stress

Proteomic analysis of Lactobacillus casei GCRL163 cell-free extracts reveals a SecB homolog and other biomarkers of prolonged heat stress

An Acid Up-Regulated Surface Protein of Lactobacillus paracasei Strain GCRL 46 is Phylogenetically Related to the Secreted Glucan- (GpbB) and Immunoglobulin-Binding (SibA) Protein of Pathogenic Streptococci

Prolonged Heat Stress of Lactobacillus paracasei GCRL163 Improves Binding to Human Colorectal Adenocarcinoma HT-29 Cells and Modulates the Relative Abundance of Secreted and Cell Surface-Located Proteins

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