Geert Schoofs

Identification of a Gene Cluster for the Biosynthesis of a Long, Galactose-Rich Exopolysaccharide in Lactobacillus rhamnosus GG and Functional Analysis of the Priming Glycosyltransferase.

ABSTRACT Cell surface polysaccharides have an established role as virulence factors in human bacterial pathogens. Less documented are the biosynthesis and biological functions of surface polysaccharides in beneficial bacteria. We identified a gene cluster that encodes the enzymes and regulatory and transporter proteins for the different steps in the biosynthesis of extracellular polysaccharides (EPS) of the well-documented probiotic strain Lactobacillus rhamnosus GG. Subsequent mutation of the welE gene, encoding the priming glycosyltransferase within this cluster, and comparative phenotypic analyses of wild-type versus mutant strains confirmed the specific function of this gene cluster in the biosynthesis of high-molecular-weight, galactose-rich heteropolymeric EPS molecules. The phenotypic analyses included monomer composition determination, estimation of the polymer length of the isolated EPS molecules, and single-molecule force spectroscopy of the surface polysaccharides. Further characterization of the welE mutant also showed that deprivation of these long, galactose-rich EPS molecules results in an increased adherence and biofilm formation capacity of L. rhamnosus GG, possibly because of less shielding of adhesins such as fimbria-like structures.

The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus

BACKGROUND The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The proteolytic core of the complex is formed by the 20S proteasome, a cylinder-shaped particle that in archaebacteria contains two different subunits (alpha and beta) and in eukaryotes contains fourteen different subunits (seven of the alpha-type and seven of the beta-type). RESULTS We have purified a 20S proteasome complex from the nocardioform actinomycete Rhodococcus sp. strain NI86/21. The complex has an apparent relative molecular mass of 690 kD, and efficiently degrades the chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of 0.05% SDS. Purified preparations reveal the existence of four subunits, two of the alpha-type and two of the beta-type, the genes for which we have cloned and sequenced. Electron micrographs show that the complex has the four-ringed, cylinder-shaped appearance typical of proteasomes. CONCLUSIONS The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri

The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

Plant Lectin-Like Bacteriocin from a Rhizosphere-Colonizing Pseudomonas Isolate

Rhizosphere isolate Pseudomonas sp. strain BW11M1, which belongs to the Pseudomonas putida cluster, secretes a heat- and protease-sensitive bacteriocin which kills P. putida GR12-2R3. The production of this bacteriocin is enhanced by DNA-damaging treatment of producer cells. We isolated a TnMod mutant of strain BW11M1 that had lost the capacity to inhibit the growth of strain GR12-2R3. A wild-type genomic fragment encompassing the transposon insertion site was shown to confer the bacteriocin phenotype when it was introduced into Escherichia coli cells. The bacteriocin structural gene was identified by defining the minimal region required for expression in E. coli. This gene was designated llpA (lectin-like putidacin) on the basis of significant homology of its 276-amino-acid product with mannose-binding lectins from monocotyledonous plants. LlpA is composed of two monocot mannose-binding lectin (MMBL) domains. Several uncharacterized bacterial genes encoding diverse proteins containing one or two MMBL domains were identified. A phylogenetic analysis of the MMBL domains present in eukaryotic and prokaryotic proteins assigned the putidacin domains to a new bacterial clade within the MMBL-containing protein family. Heterologous expression of the llpA gene also conveyed bacteriocin production to several Pseudomonas fluorescens strains. In addition, we demonstrated that strain BW11M1 and heterologous hosts secrete LlpA into the growth medium without requiring a cleavable signal sequence. Most likely, the mode of action of this lectin-like bacteriocin is different from the modes of action of previously described Pseudomonas bacteriocins.

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli - Rhodococcus shuttle vectors

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

The major secreted protein Msp1/p75 is O-glycosylated in Lactobacillus rhamnosus GG

BackgroundAlthough the occurrence, biosynthesis and possible functions of glycoproteins are increasingly documented for pathogens, glycoproteins are not yet widely described in probiotic bacteria. Nevertheless, knowledge of protein glycosylation holds important potential for better understanding specific glycan-mediated interactions of probiotics and for glycoengineering in food-grade microbes.ResultsHere, we provide evidence that the major secreted protein Msp1/p75 of the probiotic Lactobacillus rhamnosus GG is glycosylated. Msp1 was shown to stain positive with periodic-acid Schiff staining, to be susceptible to chemical deglycosylation, and to bind with the mannose-specific Concanavalin A (ConA) lectin. Recombinant expression in Escherichia coli resulted in a significant reduction in molecular mass, loss of ConA reactivity and increased sensitivity towards pronase E and proteinase K. Mass spectrometry showed that Msp1 is O- glycosylated and identified a glycopeptide TVETPSSA (amino acids 101-108) bearing hexoses presumably linked to the serine residues. Interestingly, these serine residues are not present in the homologous protein of several Lactobacillus casei strains tested, which also did not bind to ConA. The role of the glycan substitutions in known functions of Msp1 was also investigated. Glycosylation did not seem to impact significantly on the peptidoglycan hydrolase activity of Msp1. In addition, the glycan chain appeared not to be required for the activation of Akt signaling in intestinal epithelial cells by Msp1. On the other hand, examination of different cell extracts showed that Msp1 is a glycosylated protein in the supernatant, but not in the cell wall and cytosol fraction, suggesting a link between glycosylation and secretion of this protein.ConclusionsIn this study we have provided the first evidence of protein O- glycosylation in the probiotic L rhamnosus GG. The major secreted protein Msp1 is glycosylated with ConA reactive sugars at the serine residues at 106 and 107. Glycosylation is not required for the peptidoglycan hydrolase activity of Msp1 nor for Akt activation capacity in epithelial cells, but appears to be important for its stability and protection against proteases.

DISTILLER: a data integration framework to reveal condition dependency of complex regulons in Escherichia coli

We present DISTILLER, a data integration framework for the inference of transcriptional module networks. Experimental validation of predicted targets for the well-studied fumarate nitrate reductase regulator showed the effectiveness of our approach in Escherichia coli. In addition, the condition dependency and modularity of the inferred transcriptional network was studied. Surprisingly, the level of regulatory complexity seemed lower than that which would be expected from RegulonDB, indicating that complex regulatory programs tend to decrease the degree of modularity.

Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.

The proteome of Salmonella Typhimurium grown under in vivo-mimicking conditions.

To successfully infect a host, it is a prerequisite for enteric pathogens such as Salmonella enterica serovar Typhimurium to adapt to their environment, in casu the gastrointestinal tract. The adoption of an appropriate lifestyle is triggered by environmental signals such as the low oxygen availability and high osmolarity prevalent in the gut. In order to gain more insight in the changes that are induced when S. Typhimurium is adapting to these particular conditions, we used 2‐D DIGE technology to investigate the combined effect of low oxygen tension and high osmolarity on the proteome of S. Typhimurium SL1344 compared to standard laboratory conditions. As a validation of the 2‐D DIGE technique, preferential protein labeling by the Cy‐dyes was assessed and proved to be negligible. The differentially expressed proteins identified reflect very well the applied culture conditions. Furthermore, reported transcriptional changes and observed changes at the translational level show overlap. Among the metabolic processes that are upregulated under in vivo‐mimicking conditions are anaerobic fumarate respiration and the utilization of 1,2‐propanediol. We also provide evidence that S. Typhimurium expresses an arginine deiminase pathway for the catabolism of L‐arginine. The increased activity of this pathway was biochemically validated. Finally, also proteins involved in quorum sensing and virulence are differentially expressed under in vivo‐mimicking conditions. These conditions offer possibilities as a simplified model system for the host environment given the high overlap of identifications in our study and reported genuine in vivo studies, respectively.

Molecular characterization of the major outer-membrane protein OprF from plant root-colonizing Pseudomonas fluorescens

N-terminal sequence analysis of peptides generated by proteolytic treatment of the Pseudomonas fluorescens OE 28.3 major outer-membrane protein OprF, embedded in outer membranes or present in whole cells, indicated a surface-exposed location for the proline-rich region of the protein. This region is absent from the P. aeruginosa and P. syringae OprFs. Evidence was obtained for the presence of additional exposed but less accessible regions in the carboxy half of OprF. Four OprF-specific monoclonal antibodies were all directed to the C-terminal part of the protein but did not recognize a surface-exposed epitope as shown by flow cytometry. Our data support the model previously proposed for P. aeruginosa OprF in which the entire protein is embedded in the outer membrane, unlike the topology proposed for the major outer-membrane protein from Escherichia coli, OmpA, whose carboxy half resides in the periplasmic space. For six other P. fluorescens strains producing OprF proteins with different isoelectric points, the primary structure was determined by sequence analysis of the PCR-amplified oprF genes. The proline-rich domain represented the most conserved region of the different P. fluorescens OprFs. Based on the sequence of its oprF gene, it was shown that the mushroom pathogen P. tolaasii is quite closely related to P. fluorescens. Comparative sequence analysis further showed that the carboxy half of OprF contains a sequence motif that is well conserved in the enterobacterial OmpA proteins but is also present in a number of other outer-membrane proteins, including peptidoglycan-associated lipoproteins.