Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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L. lactis NZ9000 (pNZbcaP-H6) Vs. L. lactis NZ9000 (pNZ8048)

ABSTRACT: Our transcriptome data shows that the two-component system CesSR, which senses cell envelope stresses of different origins, is one of the major players when L. lactis is forced to overproduce the endogenous membrane protein BcaP, a branched-chain amino acid permease. Two-condition experiment: nisin induced overproduction of BcaP vs. control. Biological replicates: 2 controls, 2 overproduction cultures; independently grown and harvested; dye swaped on the second array.

ORGANISM(S): Lactococcus lactis subsp. cremoris NZ9000

SUBMITTER: Anne Jong

PROVIDER: E-GEOD-30776 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Previous studies have indicated that PsaR of Streptococcus pneumoniae is a manganese-dependent regulator, negatively affecting the expression of at least seven genes. Here, we extended these observations by transcriptome and proteome analysis of psaR mutants in strains D39 and TIGR4. The microarray analysis identified three shared PsaR targets: the psa-operon, pcpA, and prtA. Additionally, we found 31 genes to be regulated by PsaR in D39 only, most strikingly a cellobiose-specific PTS and a putative bacteriocin operon (sp0141-sp0146). In TIGR4, 14 PsaR gene targets were detected, with the rlrA pathogenicity islet being the most pronounced. Proteomics confirmed most of the shared gene targets. To examine the contribution of PsaR to pneumococcal virulence, we compared D39 and TIGR4 wild-type (wt) and psaR-mutants in three murine infection models. During colonization, no clear effect was observed of the psaR mutation in either D39 or TIGR4. In the pneumonia model, small but significant differences were observed in the lungs of mice infected with either D39wt or âpsaR: D39âpsaR had an initial advantage in survival in the lungs. Conversely, TIGR4âpsaR-infected mice had significantly lower bacterial loads at 24h only. Finally, during experimental bacteremia, D39âpsaR-infected mice had significantly lower bacterial loads in the blood stream than wt-infected mice for the first 24h of infection. TIGR4âpsaR showed attenuation at 36h only. In conclusion, our results show that PsaR of D39 and TIGR4 has a strain-specific role in global gene expression and in the development of bacteremia in mice. Microarray analysis was performed essentially as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). In short, 500 ml of CDM was inoculated with 10-20 colonies from agar plates, and these cultures were statically grown at 37Â°C. Samples for RNA isolation were taken when the cultures reached an optical density (OD600) of 0.2 (mid-log growth). RNA was isolated and purified using the High Pure RNA isolation kit (Roche diagnostics) as described (Hendriksen et al., 2007; Hendriksen et al., 2008a). Contaminating genomic DNA was removed by treatment with RNase-free DNase I (Roche diagnostics). RNA was isolated from three replicate cultures. Synthesis, subsequent labeling of cDNA, and microarray hybridization was performed as described (Hendriksen et al., 2007; Kloosterman et al., 2006). In all cases, dye-swapping was performed with one of the three biological replicates. Microarrays used in this study were constructed as described (Hendriksen et al., 2007; Kloosterman et al., 2006) and contain amplicons representing 2,087 ORFs of S. pneumoniae TIGR4 and 184 ORFs unique for S. pneumoniae R6, all spotted in duplicate.

Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.

Dataset Information

L. lactis NZ9000 (pNZbcaP-H6) Vs. L. lactis NZ9000 (pNZ8048)

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