Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphyloc... more Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015–2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureu...
The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highl... more The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.
Lipid-modified proteins play important roles at the interface between eubacterial cells and their... more Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gramnegative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtMdependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity. One of the most common eubacterial protein sorting (retention) signals is an amino-terminal lipid-modified cysteine residue, which anchors proteins to the cytoplasmic or outer membranes (see Refs. 1-4). The known lipid-modified proteins (lipoproteins) are involved in a large variety of processes, which range from the uptake of nutrients, resistance against antibi-N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; X-gal, 5-bromo-4chloro-3-indolyl--D-galactopyranoside.
analysis of targeted genes. in Lactococcus lactis which allows fast A system to generate chromoso... more analysis of targeted genes. in Lactococcus lactis which allows fast A system to generate chromosomal mutations
The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococca... more The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influ...
Staphylococcus aureus is a dangerous human pathogen that can cause a range of diseases. The genom... more Staphylococcus aureus is a dangerous human pathogen that can cause a range of diseases. The genome of this bacterium encodes an arsenal of virulence factors that are exported to extracytoplasmic locations. Once translocated across the membrane, these proteins are either retained at the surface of the cell or released into the environment. S. aureus contains several protein secretion pathways of which the general Sec pathway seems to be most frequently used. Other potential secretion pathways include the Twinarginine translocation (Tat), Com and Ess pathways. This study reports on the analysis of S. aureus secretion mutants. Sixteen isogenic S. aureus secretion mutants were created, and the influence of the respective mutations on the exoproteome composition was analyzed. Furthermore, the strains were tested for hemolysin production, and Caenorhabditis elegans killing. Several S. aureus mutants showed significantly altered exoproteomes, such as the secG or lgt mutants. In contrast, no obvious secretion defects were observed for the other mutant strains lacking factors required for protein translocation, precursor processing, and attachment of proteins to the cell wall. Notably, in various mutant strains second-site mutations were observed that led to the loss of hemolysin production, suggesting the acquisition of agr mutations. While this seems to be a consequence of the natural adaptive capabilities of S. aureus, it is also an important caveat for future studies on protein secretion in this organism that underscores the need for genetically stable model strains.
International Journal of Medical Microbiology, 2014
The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococ... more The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.
Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of ... more Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains W g2 and SKU. These plasmids were introduced into the plasmid-free strain L.lactis MG1363. The serine proteinases produced by these L.lactis strains were isolated, and their cleavage specificity and rate towards αs1and β-casein was investigated. The catalytic properties of both the SKU and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L.lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L.lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.
A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteri... more A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a hydrophobic peptide that is initially synthesized with an N-terminal extension. The housekeeping surface proteinase HtrA was shown to be responsible for the cleavage of precursor peptide to yield the active bacteriocin. LsbB is a relatively hydrophilic protein synthesized without an Nterminal leader sequence or signal peptide. The secretion of both polypeptides was shown to be mediated by LmrB. An L. lactis strain lacking plasmid-encoded LmrB and the chromosomally encoded LmrA is unable to secrete either of the two bacteriocins. Complementation of the strain with an active LmrB protein resulted in restored export of the two polypeptides across the cytoplasmic membrane. When expressed in an L. lactis strain that is sensitive to LsbA and LsbB, LmrB was shown to confer resistance toward both bacteriocins. It does so, most likely, by removing the two polypeptides from the cytoplasmic membrane. This is the first report in which a multidrug transporter protein is shown to be involved in both secretion and immunity of antimicrobial peptides.
Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleoti... more Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S] ؉ cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases. Lactococcus lactis is a member of the family of lactic acid Gram-positive bacteria that grow anaerobically but tolerate low concentrations of oxygen (1). Similar to all other bacteria it
The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, ... more The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr -dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.
Motivation: Genomic research laboratories need an adequate infrastructure to support management o... more Motivation: Genomic research laboratories need an adequate infrastructure to support management of their data production and research workflow. But what makes infrastructure adequate? Lack of appropriate criteria makes any decision on buying or developing a system difficult. We here report on the decision process for the case of a molecular genetics group establishing a microarray laboratory. Results: Five typical requirements for experimental genomics database systems were identified: (i) evolution ability to keep up with the fast developing genomics field, (ii) a suitable data model to deal with local diversity, (iii) suitable storage of data files in the system, (iv) easy exchange with other software, and (v) low maintenance costs. The computer scientists and the researchers of the local microarray laboratory considered alternative solutions for these five requirements and chose the following options: (i) use of automatic code generation, (ii) a customized data model based on standards, (iii) storage of datasets as black boxes instead of decomposing them in database tables, (iv) loosely linking to other programs for improved flexibility, and (v) a low-maintenance web-based user interface. The team evaluated existing microarray databases (reported in Table 1) and then decided to build a new system: Molecular Genetics Information System (MOLGENIS), implemented using code generation in a period of three months. This case can provide valuable insights and lessons to both software developers and a user community embarking on large scale genomic projects. Availability: An installation of MOLGENIS for demonstration purposes is accessible at http://molgen70.biol.rug.nl/molgenis_demo (username: guest, password: guest).
Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates ... more Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.
The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and w... more The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
... totally different regulatory responses in Bacillus subtilis. Andrzej Lulko, Henk van Noordenb... more ... totally different regulatory responses in Bacillus subtilis. Andrzej Lulko, Henk van Noordenburg, Harma Karsens, Girbe Buist, Jan Kok and Oscar Kuipers 4 Page 2. Chapter 4 64 ... T15 T30 T60 Regulon / time point of analysis Num b e ro fg enes downregulation upregulation Fig. ...
... Merchante et al., 1995; Pooley et al., 1996). Recently, a bipartite structure was detected in... more ... Merchante et al., 1995; Pooley et al., 1996). Recently, a bipartite structure was detected in the cell wall of B. subtilis using cryo-electron microscopy (Matias and Beveridge, 2005). On the extracytoplasmic side of the membrane a ...
Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathoge... more Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by met...
The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during pr... more The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells ...
Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphyloc... more Mastitis in cows is a major cause of economic losses and it is commonly associated with Staphylococcus aureus. Little is known about the S. aureus lineages causing mastitis in Mexican cattle. The aim of this study was to type S. aureus isolates causing mastitis in cows from the Comarca Lagunera region in Mexico in 2015–2016. Multi-locus variable number tandem repeat fingerprinting (MLVF) of 33 S. aureus isolates obtained from 210 milk samples revealed the MLVF clusters A (n = 1), B (n = 26), C (n = 5) and D (n = 1). Spa-typing showed that clusters A and B represent the spa-type t224, cluster C includes spa-types t3196 and t416, and cluster D represents spa-type t114. The different spa-types were mirrored by the masses of protein A bands as detected by Western blotting. Antimicrobial susceptibility testing showed that one isolate was susceptible to all antimicrobials tested, whereas all other strains were resistant only to benzylpenicillin. These findings show that only four S. aureu...
The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highl... more The C-terminal region (cA) of the major autolysin AcmA of Lactococcus lactis contains three highly similar repeated regions of 45 amino acid residues (LysM domains), which are separated by nonhomologous sequences. The cA domain could be deleted without destroying the cell wall-hydrolyzing activity of the enzyme in vitro. This AcmA derivative was capable neither of binding to lactococcal cells nor of lysing these cells while separation of the producer cells was incomplete. The cA domain and a chimeric protein consisting of cA fused to the C terminus of MSA2, a malaria parasite surface antigen, bound to lactococcal cells specifically via cA. The fusion protein also bound to many other Gram-positive bacteria. By chemical treatment of purified cell walls of L. lactis and Bacillus subtilis, peptidoglycan was identified as the cell wall component interacting with cA. Immunofluorescence studies showed that binding is on specific locations on the surface of L. lactis, Enterococcus faecalis, Streptococcus thermophilus, B. subtilis, Lactobacillus sake, and Lactobacillus casei cells. Based on these studies, we propose that LysM-type repeats bind to peptidoglycan and that binding is hindered by other cell wall constituents, resulting in localized binding of AcmA. Lipoteichoic acid is a candidate hindering component. For L. lactis SK110, it is shown that lipoteichoic acids are not uniformly distributed over the cell surface and are mainly present at sites where no MSA2cA binding is observed.
Lipid-modified proteins play important roles at the interface between eubacterial cells and their... more Lipid-modified proteins play important roles at the interface between eubacterial cells and their environment. The importance of lipoprotein processing by signal peptidase II (SPase II) is underscored by the fact that this enzyme is essential for viability of the Gramnegative eubacterium Escherichia coli. In contrast, SPase II is not essential for growth and viability of the Gram-positive eubacterium Bacillus subtilis. This could be due to alternative amino-terminal lipoprotein processing, which was shown previously to occur in SPase II mutants of B. subtilis. Alternatively, uncleaved lipoprotein precursors might be functional. To explore further the importance of lipoprotein processing in Gram-positive eubacteria, an SPase II mutant strain of Lactococcus lactis was constructed. Although some of the 39 (predicted) lactococcal lipoproteins, such as PrtM and OppA, are essential for growth in milk, the growth of SPase II mutant L. lactis cells in this medium was not affected. Furthermore, the activity of the strictly PrtMdependent extracellular protease PrtP, which is required for casein degradation, was not impaired in the absence of SPase II. Importantly, no alternative processing of pre-PrtM and pre-OppA was observed in cells lacking SPase II. Taken together, these findings show for the first time that authentic lipoprotein precursors retain biological activity. One of the most common eubacterial protein sorting (retention) signals is an amino-terminal lipid-modified cysteine residue, which anchors proteins to the cytoplasmic or outer membranes (see Refs. 1-4). The known lipid-modified proteins (lipoproteins) are involved in a large variety of processes, which range from the uptake of nutrients, resistance against antibi-N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; X-gal, 5-bromo-4chloro-3-indolyl--D-galactopyranoside.
analysis of targeted genes. in Lactococcus lactis which allows fast A system to generate chromoso... more analysis of targeted genes. in Lactococcus lactis which allows fast A system to generate chromosomal mutations
The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococca... more The autolysin AcmA of Lactococcus lactis was shown to be degraded by the extracellular lactococcal proteinase PrtP. Autolysis, as evidenced by reduction in optical density of a stationary-phase culture and concomitant release of intracellular proteins, was greatly reduced when L. lactis MG1363 cells expressed the cell wall-anchored lactococcal proteinase PrtP of the PI-type caseinolytic specificity (PI). On the other hand, lactococcal strains that did not produce the proteinase showed a high level of autolysis, which was also observed when the cells produced the secreted form of PI or a cell wall-anchored proteinase with PIII-type specificity. Autolysis was also increased when MG1363 expressed the cell wall-anchored hybrid PI/PIII-type proteinase PIac. Zymographic analysis of AcmA activity during stationary phase showed that AcmA was quickly degraded by PI and much more slowly by PrtP proteinases with PIII-type and intermediate specificities. Autolysis of L. lactis by AcmA was influ...
Staphylococcus aureus is a dangerous human pathogen that can cause a range of diseases. The genom... more Staphylococcus aureus is a dangerous human pathogen that can cause a range of diseases. The genome of this bacterium encodes an arsenal of virulence factors that are exported to extracytoplasmic locations. Once translocated across the membrane, these proteins are either retained at the surface of the cell or released into the environment. S. aureus contains several protein secretion pathways of which the general Sec pathway seems to be most frequently used. Other potential secretion pathways include the Twinarginine translocation (Tat), Com and Ess pathways. This study reports on the analysis of S. aureus secretion mutants. Sixteen isogenic S. aureus secretion mutants were created, and the influence of the respective mutations on the exoproteome composition was analyzed. Furthermore, the strains were tested for hemolysin production, and Caenorhabditis elegans killing. Several S. aureus mutants showed significantly altered exoproteomes, such as the secG or lgt mutants. In contrast, no obvious secretion defects were observed for the other mutant strains lacking factors required for protein translocation, precursor processing, and attachment of proteins to the cell wall. Notably, in various mutant strains second-site mutations were observed that led to the loss of hemolysin production, suggesting the acquisition of agr mutations. While this seems to be a consequence of the natural adaptive capabilities of S. aureus, it is also an important caveat for future studies on protein secretion in this organism that underscores the need for genetically stable model strains.
International Journal of Medical Microbiology, 2014
The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococ... more The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.
Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of ... more Plasmids containing wild-type and hybrid proteinase genes were constructed from DNA fragments of the prtP genes of Lactococcus lactis strains W g2 and SKU. These plasmids were introduced into the plasmid-free strain L.lactis MG1363. The serine proteinases produced by these L.lactis strains were isolated, and their cleavage specificity and rate towards αs1and β-casein was investigated. The catalytic properties of both the SKU and Wg2 proteinases, which differ in 44 out of 1902 amino acid residues, could be changed dramatically by the reciprocal exchange of specific fragments between the two enzymes. As a result, various L.lactis strains were constructed having new proteolytic properties that differ from those of the parental strains. Furthermore, two segments in the proteinase could be identified that contribute significantly to the cleavage specificity towards casein; within these two segments, several amino acid residues were identified that are important for substrate cleavage rate and specificity. The results also indicate that the lactococcal proteinase has an additional domain involved in substrate binding compared with the related subtilisins. This suggests that the 200 kd L.lactis proteinase may be the representative of a new subclass of subtilisin-like enzymes.
A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteri... more A natural isolate of Lactococcus lactis was shown to produce two narrow spectrum class II bacteriocins, designated LsbA and LsbB. The cognate genes are located on a 5.6-kb plasmid within a gene cluster specifying LmrB, an ATP-binding cassette-type multidrug resistance transporter protein. LsbA is a hydrophobic peptide that is initially synthesized with an N-terminal extension. The housekeeping surface proteinase HtrA was shown to be responsible for the cleavage of precursor peptide to yield the active bacteriocin. LsbB is a relatively hydrophilic protein synthesized without an Nterminal leader sequence or signal peptide. The secretion of both polypeptides was shown to be mediated by LmrB. An L. lactis strain lacking plasmid-encoded LmrB and the chromosomally encoded LmrA is unable to secrete either of the two bacteriocins. Complementation of the strain with an active LmrB protein resulted in restored export of the two polypeptides across the cytoplasmic membrane. When expressed in an L. lactis strain that is sensitive to LsbA and LsbB, LmrB was shown to confer resistance toward both bacteriocins. It does so, most likely, by removing the two polypeptides from the cytoplasmic membrane. This is the first report in which a multidrug transporter protein is shown to be involved in both secretion and immunity of antimicrobial peptides.
Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleoti... more Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S] ؉ cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases. Lactococcus lactis is a member of the family of lactic acid Gram-positive bacteria that grow anaerobically but tolerate low concentrations of oxygen (1). Similar to all other bacteria it
The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, ... more The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr -dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.
Motivation: Genomic research laboratories need an adequate infrastructure to support management o... more Motivation: Genomic research laboratories need an adequate infrastructure to support management of their data production and research workflow. But what makes infrastructure adequate? Lack of appropriate criteria makes any decision on buying or developing a system difficult. We here report on the decision process for the case of a molecular genetics group establishing a microarray laboratory. Results: Five typical requirements for experimental genomics database systems were identified: (i) evolution ability to keep up with the fast developing genomics field, (ii) a suitable data model to deal with local diversity, (iii) suitable storage of data files in the system, (iv) easy exchange with other software, and (v) low maintenance costs. The computer scientists and the researchers of the local microarray laboratory considered alternative solutions for these five requirements and chose the following options: (i) use of automatic code generation, (ii) a customized data model based on standards, (iii) storage of datasets as black boxes instead of decomposing them in database tables, (iv) loosely linking to other programs for improved flexibility, and (v) a low-maintenance web-based user interface. The team evaluated existing microarray databases (reported in Table 1) and then decided to build a new system: Molecular Genetics Information System (MOLGENIS), implemented using code generation in a period of three months. This case can provide valuable insights and lessons to both software developers and a user community embarking on large scale genomic projects. Availability: An installation of MOLGENIS for demonstration purposes is accessible at http://molgen70.biol.rug.nl/molgenis_demo (username: guest, password: guest).
Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates ... more Lactococcus lactis IL1403 harbors a putative sortase A (SrtA) and 11 putative sortase substrates that carry the canonical LPXTG signature of such substrates. We report here on the functionality of SrtA to anchor five LPXTG substrates to the cell wall, thus suggesting that SrtA is the housekeeping sortase in L. lactis IL1403.
The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and w... more The opportunistic pathogen Staphylococcus aureus has become a major threat for human health and well-being by developing resistance to antibiotics and by fast evolution into new lineages that rapidly spread within the healthy human population. This calls for development of active or passive immunization strategies to prevent or treat acute phase infections. Since no such anti-staphylococcal immunization approaches are available for clinical implementation, the present studies were aimed at identifying new leads for their development. For this purpose, we profiled the cell-surface-exposed staphylococcal proteome under infection-mimicking conditions by combining two approaches for "bacterial shaving" with immobilized or soluble trypsin and subsequent mass spectrometry analysis of liberated peptides. In parallel, non-covalently cell-wall-bound proteins extracted with potassium thiocyanate and the exoproteome fraction were analyzed by gel-free proteomics. All data are available through ProteomeXchange accession PXD000156. To pinpoint immunodominant bacterial-surface-exposed epitopes, we screened selected cell-wall-attached proteins of S. aureus for binding of immunoglobulin G from patients who have been challenged by different types of S. aureus due to chronic wound colonization. The combined results of these analyses highlight particular cell-surface-exposed S. aureus proteins with highly immunogenic exposed epitopes as potential targets for development of protective anti-staphylococcal immunization strategies.
Take-down policy If you believe that this document breaches copyright please contact us providing... more Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
... totally different regulatory responses in Bacillus subtilis. Andrzej Lulko, Henk van Noordenb... more ... totally different regulatory responses in Bacillus subtilis. Andrzej Lulko, Henk van Noordenburg, Harma Karsens, Girbe Buist, Jan Kok and Oscar Kuipers 4 Page 2. Chapter 4 64 ... T15 T30 T60 Regulon / time point of analysis Num b e ro fg enes downregulation upregulation Fig. ...
... Merchante et al., 1995; Pooley et al., 1996). Recently, a bipartite structure was detected in... more ... Merchante et al., 1995; Pooley et al., 1996). Recently, a bipartite structure was detected in the cell wall of B. subtilis using cryo-electron microscopy (Matias and Beveridge, 2005). On the extracytoplasmic side of the membrane a ...
Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathoge... more Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by met...
The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during pr... more The optical density of a culture of lactococcus lactis MG1363 was reduced more than 60% during prolonged stationary phase. Reduction in optical density (autolysis) was almost absent in a culture of an isogenic mutant containing a deletion in the major autolysin gene, acmA. An acmA mutant carrying multiple coples of a plasmid encoding AcmA lysed to a greater extent than the wild-type strain did. Intercellular action of AcmA was shown by mixing end-exponential-phase cultures of an acmA deletion mutant and a tripeptidase (pepT) deletion mutant. PepT, produced by the acmA mutant, was detected in the supernatant of the mixed culture, but no PepT was present in the culture supernatant of the acmA mutant. A plasmid was constructed in which acmA, lacking its own promoter, was placed downstream of the inducible promoter/operator region of the temperate lactococcal bacteriophage r1t. After mitomycin induction of an exponential-phase culture of L. lactis LL302 carrying this plasmid, the cells ...
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