Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster
Tarazanova, Mariya ; Beerthuyzen, M. ; Siezen, Roland ; Fernandez Gutierrez, Maria ; Jong, Anne de; Meulen, Sjoerd van der; Kok, Jan ; Bachmann, Herwig - \ 2016
PLoS ONE 11 (2016)12. - ISSN 1932-6203
Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmidfree and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.
Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus
Sieuwerts, S. ; Molenaar, D. ; Hijum, S.A.F.T. van; Beerthuyzen, M. ; Stevens, M.J.A. ; Janssen, P.W. ; Ingham, C.J. ; Bok, F.A.M. de; Vos, W.M. de; Hylckama Vlieg, J.E.T. van - \ 2010
Applied and Environmental Microbiology 76 (2010)33. - ISSN 0099-2240 - p. 7775 - 7784.
lactic-acid bacteria - microarray data - milk - metabolism - plantarum - delbrueckii - pathways - yogurt - identification - proteinases
Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process
Population Heterogeneity of Lactobacillus plantarum WCFS1 Microcolonies in Response to and Recovery from Acid Stress
Ingham, C.J. ; Beerthuyzen, M. ; Vlieg, J.E.T.V.H. - \ 2008
Applied and Environmental Microbiology 74 (2008)24. - ISSN 0099-2240 - p. 7750 - 7758.
individual bacterial-cells - lag times - listeria-monocytogenes - antibiotic tolerance - escherichia-coli - bacillus-cereus - image-analysis - low ph - growth - filamentation
Within an isogenic microbial population in a homogenous environment, individual bacteria can still exhibit differences in phenotype. Phenotypic heterogeneity can facilitate the survival of subpopulations under stress. As the gram-positive bacterium Lactobacillus plantarum grows, it acidifies the growth medium to a low pH. We have examined the growth of L. plantarum microcolonies after rapid pH downshift (pH 2 to 4), which prevents growth in liquid culture. This acidification was achieved by transferring cells from liquid broth onto a porous ceramic support, placed on a base of low-pH MRS medium solidified using Gelrite. We found a subpopulation of cells that displayed phenotypic heterogeneity and continued to grow at pH 3, which resulted in microcolonies dominated by viable but elongated (filamentous) cells lacking septation, as determined by scanning electron microscopy and staining cell membranes with the lipophilic dye FM4-64. Recovery of pH-stressed cells from these colonies was studied by inoculation onto MRS- Gelrite-covered slides at pH 6.5, and outgrowth was monitored by microscopy. The heterogeneity of the population, calculated from the microcolony areas, decreased with recovery from pH 3 over a period of a few hours. Filamentous cells did not have an advantage in outgrowth during recovery. Specific regions within single filamentous cells were more able to form rapidly dividing cells, i.e., there was heterogeneity even within single recovering cells.
Two homologous Agr-like quorum-sensing systems cooperatively control adherence, cell morphology, and cell viability properties in Lactobacillus plantarum WCFS1
Fujii, T. ; Ingham, C.J. ; Nakayama, J. ; Beerthuyzen, M.M. ; Kunuki, R. ; Molenaar, D. ; Sturme, M.H.J. ; Vaughan, E.E. ; Kleerebezem, M. ; Vos, W.M. de - \ 2008
Journal of Bacteriology 190 (2008)23. - ISSN 0021-9193 - p. 7655 - 7665.
gram-positive bacteria - staphylococcus-aureus - bacillus-subtilis - biofilm formation - escherichia-coli - transcriptional regulators - enterococcus-faecalis - streptococcus-mutans - signal-transduction - lactococcus-lactis
A two-component regulatory system of Lactobacillus plantarum, encoded by genes designated lamK and lamR (hpk10 and rrp10), was studied. The lamK and lamR genes encode proteins which are highly homologous to the quorum-sensing histidine kinase LamC and the response regulator LamA, respectively. Transcription analysis of the lamKR operon and the lamBDCA operon and liquid chromatography-mass spectrometry analysis of production of the LamD558 autoinducing peptide were performed for DeltalamA, DeltalamR, DeltalamA DeltalamR deletion mutants and a wild-type strain. The results suggested that lamA and lamR are cooperating genes. In addition, typical phenotypes of the DeltalamA mutant, such as reduced adherence to glass surfaces and filamentous cell morphology, were enhanced in the DeltalamA DeltalamR mutant. Microarray analysis suggested that the same cell wall polysaccharide synthesis genes, stress response-related genes, and cell wall protein-encoding genes were affected in the DeltalamA and DeltalamA DeltalamR mutants. However, the regulation ratio was more significant for the DeltalamA DeltalamR mutant, indicating the cooperative effect of LamA and LamR
Quantitative analysis of population heterogeneity of the adaptive salt stress response and growth capacity of Bacillus cereus ATCC 14579
Besten, H.M.W. den; Ingham, C.J. ; Hylckama Vlieg, J.E.T. van; Beerthuyzen, M.M. ; Zwietering, M.H. ; Abee, T. - \ 2007
Applied and Environmental Microbiology 73 (2007)15. - ISSN 0099-2240 - p. 4797 - 4804.
escherichia-coli o157-h7 - listeria-monocytogenes - lag times - single-cell - bacterial-populations - clostridium-botulinum - individual cells - image-analysis - inoculum size - heat
Bacterial populations can display heterogeneity with respect to both the adaptive stress response and growth capacity of individual cells. The growth dynamics of Bacillus cereus ATCC 14579 during mild and severe salt stress exposure were investigated for the population as a whole in liquid culture. To quantitatively assess the population heterogeneity of the stress response and growth capacity at a single-cell level, a direct imaging method was applied to monitor cells from the initial inoculum to the microcolony stage. Highly porous Anopore strips were used as a support for the culturing and imaging of microcolonies at different time points. The growth kinetics of cells grown in liquid culture were comparable to those of microcolonies grown upon Anopore strips, even in the presence of mild and severe salt stress. Exposure to mild salt stress resulted in growth that was characterized by a remarkably low variability of microcolony sizes, and the distributions of the log10-transformed microcolony areas could be fitted by the normal distribution. Under severe salt stress conditions, the microcolony sizes were highly heterogeneous, and this was apparently caused by the presence of both a nongrowing and growing population. After discriminating these two subpopulations, it was shown that the variability of microcolony sizes of the growing population was comparable to that of non-salt-stressed and mildly salt-stressed populations. Quantification of population heterogeneity during stress exposure may contribute to an optimized application of preservation factors for controlling growth of spoilage and pathogenic bacteria to ensure the quality and safety of minimally processed foods.
Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose biosynthesis
Boels, I.C. ; Beerthuyzen, M.M. ; Kosters, M.H. ; Kaauwen, M.P.W. van; Kleerebezem, M. ; Vos, W.M. de - \ 2004
Journal of Bacteriology 186 (2004)5. - ISSN 0021-9193 - p. 1239 - 1248.
controlled gene-expression - gram-positive bacteria - subsp cremoris - capsular polysaccharide - streptococcus-pneumoniae - acid bacteria - cell-wall - o-antigen - exopolysaccharide production - escherichia-coli
dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.
Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity
Ra, R. ; Beerthuyzen, M.M. ; Vos, W.M. de; Saris, P.E.J. ; Kuipers, O.P. - \ 1999
Microbiology 145 (1999). - ISSN 1350-0872 - p. 1227 - 1233.
Functional analysis of promoters in the gene cluster of Lactococcus lactis.
Ruyter, P.G.G.A. de; Kuipers, O.P. ; Beerthuyzen, M.M. ; Alen-Boerrigter, I.J. van; Vos, W.M. de - \ 1996
Journal of Bacteriology 178 (1996). - ISSN 0021-9193 - p. 3434 - 3439.
Protein engineering and biosynthesis of nisin and regulation of transcription of the structural nisA gene.
Kuipers, O.P. ; Rollema, H.S. ; Beerthuyzen, M.M. ; Siezen, R.J. ; Vos, W.M. de - \ 1995
International Dairy Journal 5 (1995). - ISSN 0958-6946 - p. 785 - 795.
|Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction.
Kuipers, O.P. ; Beerthuyzen, M.M. ; Ruyter, P.G.G.A. de; Luesink, E.J. ; Vos, W.M. de - \ 1995
Journal of Biological Chemistry 270 (1995). - ISSN 0021-9258 - p. 27299 - 27304.
|Biosynthesis and engineering of the antimicrobial peptide nisin of Lactocococus lactis.
Siezen, R.J. ; Rollema, H.S. ; Beerthuyzen, M.M. ; Meer, J.R. van der; Vos, W.M. de; Kuipers, O.P. - \ 1995
In: Protein engineering and complementary technologies / Geisow, M.J., Epton, R., - p. 74 - 77.
|Genetics of the nisin operon and the sucrose-nisin conjugative transposon Tn5276.
Vos, W.M. de; Beerthuyzen, M.M. ; Luesink, E.L. ; Kuipers, O.P. - \ 1995
Developments in biological standardization 85 (1995). - ISSN 0301-5149 - p. 617 - 626.
|Genetics of the nisin operon and the sucrose nisin transposon Tn5276.
Vos, W.M. de; Rauch, P.J.G. ; Beerthuyzen, M.M. ; Kuipers, O.P. - \ 1994
In: Abstract 4th ASM Conf. Genetics of Streptococcal genetics. Santa Fe, USA (1994)
|Control of nisin gene expression.
Vos, W.M. de; Beerthuyzen, M. ; Ruyter, P. de; Lueseink, E. ; Kuipers, O.P. - \ 1994
In: Abstract 2nd Workshop Lantiobiotics. Papendal, NL - p. 21 - 21.
Distribution and evolution of nisin sucrose elements in Lactococcus lactis.
Rauch, P.J.G. ; Beerthuyzen, M. ; Vos, W.M. de - \ 1994
Applied and Environmental Microbiology 60 (1994)6. - ISSN 0099-2240 - p. 1798 - 1804.
The distribution, architecture, and conjugal capacity of nisin-sucrose elements in wild-type Lactococcus lactis strains were studied. Element architecture was analyzed with the aid of hybridizations to different probes derived from the nisin-sucrose transposon Tn5276 of L. lactis NIZO R5, including its left and right ends, the nisA gene, and IS1068 (previously designated iso-IS904), located between the left end and the nisA gene. Three classes of nisin-sucrose elements could be distinguished in the 13 strains investigated. Classes I and II consist of conjugative transposons containing a nisA gene and a nisZ gene, respectively. Representative conjugative transposons of these classes include Tn5276 (class I) from L. lactis NIZO R5 and Tn5278 (class II) from L. lactis ILC11. The class II transposon found in L. lactis NCK400 and probably all class II elements are devoid of IS1068-like elements, which eliminates the involvement of an iso-IS1068 element in conjugative transposition. Members of class III contain a nisZ gene, are nonconjugative, and do not contain sequences similar to the left end of Tn5276 at the appropriate position. The class III element from L. lactis NIZO 22186 was found to contain an iso-IS1068 element, termed IS1069, at a position corresponding to that of IS1068 in Tn5276 but in the inverted orientation. The results suggest that an iso-IS1068-mediated rearrangement is responsible for the dislocation of the transposon's left end in this strain. A model for the evolution of nisin-sucrose elements is proposed, and the practical implications for transferring nisin A or nisin Z production and immunity are discussed.
|Biosynthesis of the antimicrobial peptide nisin: structure, organization and function of nisin operon genes.
Kuipers, O.P. ; Beerthuyzen, M.M. ; Meer, J.R. van der; Siezen, R.J. ; Vos, W.M. de - \ 1994
In: Abstract 5th Neth. Congr. Biotechnology. Neth. Biotechnol. Soc. Amsterdam (1994) PK-19
|Biosynthesis and protein engineering of nisin.
Kuipers, O.P. ; Rollema, H.S. ; Beerthuyzen, M.M. ; Meer, J.R. van der; Vos, W.M. de; Siezen, R.J. - \ 1994
In: Proc. 6th Eur. Congr. Biotechnology, L. Alberghina et al. (eds.). Elsevier Sci. Publ - p. 441 - 446.
Characterization of the Lactococcus lactis nisin A operon genes nisP encoding a subtilisin-like serine protease involved in processing and nisR encoding a regulatory protein involved in nisin biosynthesis.
Meer, J.R. van der; Polman, J. ; Beerthuyzen, M.M. ; Siezen, R.J. ; Kuipers, O.P. ; Vos, W.M. de - \ 1993
Journal of Bacteriology 175 (1993). - ISSN 0021-9193 - p. 2578 - 2588.