Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus
Mohedano, M.L. ; Garcia-Cayuela, T. ; Perez-Ramos, A. ; Gaiser, R.A. ; Requena, T. ; Lopez, P. - \ 2015
Journal of Industrial Microbiology and Biotechnology 42 (2015)2. - ISSN 1367-5435 - p. 247 - 253.
lactic-acid bacteria - controlled gene-expression - streptococcus-pneumoniae - lactococcus-lactis - plasmid - cloning
Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.
H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase
Hertzberger, R. ; Arents, J. ; Dekker, H.L. ; Pridmore, R.D. ; Gysler, C. ; Kleerebezem, M. ; Mattos, M.J.T. de - \ 2014
Applied and Environmental Microbiology 80 (2014)7. - ISSN 0099-2240 - p. 2229 - 2239.
hydrogen-peroxide production - alkyl hydroperoxide reductase - activated-receptor-gamma - lactic-acid bacteria - escherichia-coli - streptococcus-pneumoniae - amphibacillus-xylanus - pseudomonas-putida - johnsonii ncc-533 - crystal-structure
Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The K-m for FMN is 30 +/- 8 mu M, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii.
Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation
Bokhorst-van de Veen, H. van; Bongers, R.S. ; Wels, M. ; Bron, P.A. ; Kleerebezem, M. - \ 2013
Microbial Cell Factories 12 (2013)1. - ISSN 1475-2859 - 15 p.
gram-positive bacteria - heat-shock response - lactic-acid bacteria - bacillus-subtilis - listeria-monocytogenes - gastrointestinal-tract - low gc - streptococcus-pneumoniae - comparative genomics - helicobacter-pylori
Background - To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. Results - While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33¿±¿0.02 h-1 and 0.34¿±¿0.03 h-1, respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. Conclusions - Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium
WalK, the path towards new antibacterials with low potential for resistance development
Velikova, N.R. ; Bem, A.E. ; Baarlen, P. van; Wells, J. ; Marina, A. - \ 2013
ACS Medicical Chemistry Letters 4 (2013)10. - ISSN 1948-5875 - p. 891 - 894.
streptococcus-pneumoniae - signal-transduction - histidine kinase - discovery - inhibitors - system - genes
Resistance to antibiotics used in the treatment of bacterial infectious diseases is a global health problem. More than a decade ago, two-component systems such as WalKR were proposed as ideal targets for the development of new antibiotics. Biochemical screens for WalKR inhibitors using compound libraries have identified many hits, some of which were shown to have non-specific effects. The recently published structures of the S. mutans and B. subtilis WalK provide the opportunity to study inhibitors of WalK autophosphorylation at the atomic level and means to design compounds with improved specificity and affinity using a structure-based approach.
Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli
García-Cayuela, T. ; Cadiñanos, L.P. de; Mohedano, M.L. ; Palencia, P.F. de; Boden, D. ; Wells, J. ; Peláez, C. ; López, P. ; Requena, T. - \ 2012
Applied Microbiology and Biotechnology 96 (2012)1. - ISSN 0175-7598 - p. 171 - 181.
lactococcus-lactis - streptococcus-pneumoniae - genetic tools - in-vitro - green - cremoris - red - transformation - expression - faecalis
Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.
Inflammation in the middle ear of children with recurrent or chronic otitis media is associated with bacterial load
Stol, K. ; Diavatopoulos, D.A. ; Graamans, K. ; Engel, J.A. ; Melchers, W.J.G. ; Savelkoul, H.F.J. ; Hays, J.P. ; Warris, A. ; Hermans, P.W.M. - \ 2012
The Pediatric Infectious Disease Journal 31 (2012)11. - ISSN 0891-3668 - p. 1128 - 1134.
respiratory-syncytial-virus - real-time pcr - streptococcus-pneumoniae - haemophilus-influenzae - moraxella-catarrhalis - immunoregulatory cytokines - effusion - rhinovirus - infections - expression
Background: Viral upper respiratory tract infections have been described as an important factor in the development of otitis media (OM), although it is unclear whether they facilitate bacterial OM or can directly cause OM. To clarify the role of viral infections in OM, we compared the relative contribution of viruses and bacteria with the induction of inflammatory cytokine responses in the middle ear of children suffering from OM. Methods: Children up to 5 years of age, with recurrent or chronic episodes of OM and scheduled for ventilation tube insertion were enrolled in a prospective study. Middle ear fluids (n = 116) were collected during surgery, and quantitative polymerase chain reaction was performed to detect bacterial and viral otopathogens, that is, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and 15 respiratory viruses. Finally, concentrations of the inflammatory mediators interleukin (IL)-1 beta, IL-6, IL-8, IL-10, IL-17a and tumor necrosis factor-a were determined. Results: Middle ear fluids were clustered into 4 groups, based on the detection of viruses (28%), bacteria (27%), both bacteria and viruses (27%) or no otopathogens (19%). Bacterial detection was associated with significantly elevated concentrations of cytokines compared with middle ear fluids without bacteria (P <0.001 for all cytokines tested) in a bacterial load-dependent and species-dependent manner. In contrast, the presence of viruses was not associated with changes in cytokine values, and no synergistic effect between viral-bacterial coinfections was observed. Conclusions: The presence of bacteria, but not viruses, is associated with an increased inflammatory response in the middle ear of children with recurrent or chronic OM.
Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
Remus, D.M. ; Kranenburg, R. van; Swam, I.I. van; Taverne, N. ; Bongers, R.S. ; Wels, M. ; Wells, J. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
Microbial Cell Factories 11 (2012)1. - ISSN 1475-2859 - 10 p.
lactic-acid bacteria - gene-expression omnibus - streptococcus-pneumoniae - lactococcus-lactis - exopolysaccharide biosynthesis - microarray data - subsp cremoris - rhamnosus gg - identification - strains
Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results - The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The ¿cps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-¿B activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions - Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.
beta-Lactam and glycopeptide antibiotics: first and last line of defense?
Jovetic, S. ; Yang Zhu, Yang ; Marcone, G.L. ; Marinelli, F. ; Tramper, J. - \ 2010
Trends in Biotechnology 28 (2010)12. - ISSN 0167-7799 - p. 596 - 604.
resistant staphylococcus-aureus - methicillin resistance - antimicrobial resistance - lipid-ii - streptococcus-pneumoniae - vancomycin resistance - crystal-structures - biological cost - united-states - fitness cost
Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics beta-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies Several strategies have enriched the pipeline of bacterial cell-wall inhibitors examples include combining screening strategies with lesser explored microbial diversity, or reinventing known scaffolds based on structure-function relationships Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance
The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel a-glucosides through reverse phosphorolysis by maltose phosphorylase
Nakai, H. ; Baumann, M.J. ; Petersen, B.O. ; Westphal, Y. ; Schols, H.A. ; Dilokpimol, A. ; Hachem, M.A. ; Lathinen, S.J. ; Duus, J.O. ; Svensson, B. - \ 2009
FEBS Journal 276 (2009). - ISSN 1742-464X - p. 7353 - 7365.
escherichia-coli - bacillus-subtilis - crystal-structure - thermoanaerobacter-brockii - trehalose phosphorylase - streptococcus-pneumoniae - cellobiose phosphorylase - nucleotide-sequence - lactococcus-lactis - cloned gene
A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing ß-glucose 1-phosphate (ß-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and ß-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mmß-Glc 1-P and 50 mm glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or l-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be a-Glcp-(1¿4)-Glcp (maltose), a-Glcp-(1¿4)-GlcNp (maltosamine), a-Glcp-(1¿4)-GlcNAcp (N-acetyl maltosamine), a-Glcp-(1¿4)-Manp, a-Glcp-(1¿4)-Xylp and a-Glcp-(1¿4)- l-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.
Heterologous expression of the pneumococcal serotype 14 polysaccharide in Lactococcus lactis requires lactococcal epsABC regulatory genes
Nierop Groot, M.N. ; Godefrooij, J. ; Kleerebezem, M. - \ 2008
Applied and Environmental Microbiology 74 (2008)3. - ISSN 0099-2240 - p. 912 - 915.
streptococcus-pneumoniae - capsular polysaccharide - cell-wall - exopolysaccharides - length - cpsd
The pneumococcal serotype 14 polysaccharide was produced in Lactococcus lactis by coexpressing pneumococcal polysaccharide type 14-specific genes (cpsFGHIJKL(14)) with the lactococcal regulatory and priming glucosyltransferase-encoding genes specific for B40 polysaccharide (epsABCD(B40)). The polysaccharide produced by Lactococcus was secreted in the medium, simplifying downstream processing and polysaccharide isolation from culture broth
Mucosal delivery of a pneumococcal vaccine using lactococcus lactis affords protection against respiratory infection
Hanniffy, S.B. ; Carter, A.T. ; Hitchin, E. ; Wells, J. - \ 2007
The Journal of Infectious Diseases 195 (2007)2. - ISSN 0022-1899 - p. 185 - 193.
surface protein-a - human alveolar macrophages - cellular immune-responses - toxin fragment-c - streptococcus-pneumoniae - acid bacteria - conjugate vaccines - oral immunization - heterologous pspa - lung infection
Background - Economical and effective vaccines against Streptococcus pneumoniae (pneumococcus) are needed for implementation in poorer countries where the disease burden is highest. Here, we evaluated Lactococcus lactis intracellularly producing the pneumococcal surface protein A (PspA) as a mucosal vaccine in conferring protection against pneumococcal disease. Methods - Mice were intranasally (inl) immunized with the lactococcal vaccine. Control groups were also immunized with similar amounts of recombinant PspA administered inl or subcutaneously with alum. PspA-specific antibodies in serum samples and lung lavage fluids were measured before challenge in intraperitoneal sepsis and inl respiratory-infection models of pneumococcal disease. Results - The lactococcal vaccine afforded better protection against respiratory challenge with pneumococcus than did vaccination with purified antigen given inl or by injection with alum. This finding was associated with a shift toward a Th1-mediated immune response characterized by reduced antibody titers to the PspA antigen. In the sepsis model, the lactococcal vaccine afforded resistance to disease on a par with that obtained with the injected vaccine, demonstrating its efficacy against different forms of pneumococcal disease. Conclusion - Given the safety profile of L. lactis, there is considerable potential to develop a pneumococcal vaccine for use in humans and to broaden this approach to combat other major pathogens
Comparative analysis of two-component signal transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis
Been, M.W.H.J. de; Francke, C. ; Moezelaar, R. ; Abee, T. ; Siezen, R.J. - \ 2006
Microbiology 152 (2006)10. - ISSN 1350-0872 - p. 3035 - 3048.
multiple sequence alignment - beta-lactam resistance - staphylococcus-aureus - regulatory system - streptococcus-pneumoniae - gene-expression - listeria-monocytogenes - escherichia-coli - protein-kinase - multistep phosphorelay
Members of the Bacillus cereus group are ubiquitously present in the environment and can adapt to a wide range of environmental fluctuations. In bacteria, these adaptive responses are generally mediated by two-component signal transduction systems (TCSs), which consist of a histidine kinase (HK) and its cognate response regulator (RR). With the use of in silico techniques, a complete set of HKs and RRs was recovered from eight completely sequenced B. cereus group genomes. By applying a bidirectional best-hits method combined with gene neighbourhood analysis, a footprint of these proteins was made. Around 40 HK-RR gene pairs were detected in each member of the B. cereus group. In addition, each member contained many HK and RR genes not encoded in pairs (`orphans¿). Classification of HKs and RRs based on their enzymic domains together with the analysis of two neighbour-joining trees of these domains revealed putative interaction partners for most of the `orphans¿. Putative biological functions, including involvement in virulence and host¿microbe interactions, were predicted for the B. cereus group HKs and RRs by comparing them with those of B. subtilis and other micro-organisms. Remarkably, B. anthracis appeared to lack specific HKs and RRs and was found to contain many truncated, putatively non-functional, HK and RR genes. It is hypothesized that specialization of B. anthracis as a pathogen could have reduced the range of environmental stimuli to which it is exposed. This may have rendered some of its TCSs obsolete, ultimately resulting in the deletion of some HK and RR genes
Genome-based in silico detection of putative manganese transport systems in Lactobacillus plantarum and their genetic analysis
Groot, M.N. ; Klaassens, E.S. ; Vos, W.M. de; Delcour, J. ; Hols, P. ; Kleerebezem, M. - \ 2005
Microbiology 151 (2005)4. - ISSN 1350-0872 - p. 1229 - 1238.
p-type atpase - lactococcus-lactis - streptococcus-pneumoniae - escherichia-coli - radiation-resistance - nucleotide-sequence - molecular-cloning - abc transporter - nramp proteins - expression
Manganese serves an important function in Lactobacillus plantarum in protection against oxidative stress and this bacterium can accumulate Mn2+ up to millimolar levels intracellularly. Although the physiological role of Mn2+ and the uptake of this metal ion have been well documented, the only uptake system described so far for this bacterium is the Mn2+- and Cd2+-specific P-type ATPase (MntA). Recently, the genome of L. plantarum WCFS1 has been sequenced allowing in silico detection of genes potentially encoding Mn2+ transport systems, using established microbial Mn2+ transporters as the query sequence. This genome analysis revealed that L. plantarum WCFS1 encodes, besides the previously described mntA gene, an ABC transport system (mtsCBA) and three genes encoding Nramp transporters (mntH1, mntH2 and mntH3). The expression of three (mtsCBA, mntH1 and mntH2) of the five transport systems was specifically derepressed or induced upon Mn2+ limitation, supporting their role in Mn2+ homeostasis in L. plantarum. However, in contrast to previous reports, mntA expression remains below detection levels in both Northern and real-time RT-PCR analysis in both Mn2+ excess and starvation conditions. Growth of WCFS1 derivatives mutated in mntA, mtsA or mntH2, or both mtsA and mntH2 appears unaffected under Mn2+ excess or Mn2+ limitation. Moreover, intracellular Mn2+ concentrations remained unaltered in these mutants compared to the wild-type. This may suggest that this species is highly adaptive in response to inactivation of these genes or, alternatively, that other transporters that have not yet been identified as Mn2+ transporters in bacteria are involved in Mn2+ homeostasis in L. plantarum
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.
Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: Strain diversity, host range, and recombination
Schouls, L.M. ; Reulen, S. ; Duim, B. ; Wagenaar, J.A. ; Willems, R.J.L. ; Dingle, K.E. ; Colles, F.M. ; Embden, J.D.A. van - \ 2003
Journal of Clinical Microbiology 41 (2003)1. - ISSN 0095-1137 - p. 15 - 26.
guillain-barre - mycobacterium-tuberculosis - streptococcus-pneumoniae - genome sequence - gastroenteritis - epidemiology - netherlands - outbreak - poultry
Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.
High-throughput PCR Screening of Genes for Three-component Regulatory System Putatively involved in Quorum Sensing from Low-G+C Gram-positive Bacteria
Nakayama, J. ; Akkermans, A.D.L. ; Vos, W.M. de - \ 2003
Bioscience, Biotechnology and Biochemistry 67 (2003)3. - ISSN 0916-8451 - p. 480 - 489.
biosynthesis-activating pheromone - 2-component signal-transduction - lactobacillus-plantarum c-11 - complete genome sequence - gram-positive bacteria - streptococcus-pneumoniae - enterococcus-faecalis - bacillus-subtilis - staphylococcus-aureus - competence-phero
Quorum sensing of Gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.