Biomedical subjects

Philip M Giffard

Publications and source records attributed to Philip M Giffard.

At least 19 recordsLinked to original sources

PubMed2006

Lactobacillus fermentum BR11, a potential new probiotic, alleviates symptoms of colitis induced by dextran sulfate sodium (DSS) in rats.

Current treatments for inflammatory bowel disease (IBD) are relatively ineffective. Recently, probiotics have emerged as a potential treatment modality for numerous gastrointestinal disorders, including IBD. Few probiotics, however, have undergone appropriate preclinical screening in vivo. The current study compared the effects of four candidate probiotics on development of dextran sulfate sodium (DSS)-induced colitis in rats. Sprague Dawley rats were gavaged 1 mL of the potential probiotic (1 x 10(10) CFU/mL), or vehicle, twice daily for 14 days. Strains tested were Lactobacillus rhamnosus GG (LGG), Streptococcus thermophilus TH-4 (TH-4), Bifidobacterium lactis Bb12 (Bb12) and Lactobacillus fermentum BR11 (BR11). Colitis was induced from day 7 to 14 via administration of 2% DSS in drinking water. Disease activity index (DAI) was monitored daily until rats were killed at day 14. DAI decreased in DSS+Bb12 and DSS+BR11 compared to DSS+Vehicle. Colon length increased in DSS+BR11 (10%) and DSS+LGG (10%) compared to DSS+Vehicle. DSS+Bb12 and DSS+BR11 prevented the distal colon crypt hyperplasia evident in DSS+Vehicle, DSS+LGG and DSS+TH-4. BR11 was most effective at reducing colitic symptoms. Bb12 had minimal effects, whilst TH-4 did not prevent DSS-colitis and LGG actually exacerbated some indicators of colitis. Further studies into the potential benefits of L. fermentum BR11 are indicated.

Animals↗

PubMed2006

Inhibition of Staphylococcus aureus growth on tellurite-containing media by Lactobacillus reuteri Is dependent on CyuC and thiol production.

Lactobacillus reuteri inhibits Staphylococcus aureus growth on Baird-Parker agar. This activity required the presence of tellurite and was not shared with other lactic acid bacteria or an L. reuteri mutant defective in cystine metabolism. Secreted products generated from L. reuteri cystine metabolism and thiols were shown to augment tellurite toxicity.

Agar↗

PubMed2006

Fingerprinting of Campylobacter jejuni by using resolution-optimized binary gene targets derived from comparative genome hybridization studies.

The aim of this investigation was to exploit the vast comparative data generated by comparative genome hybridization (CGH) studies of Campylobacter jejuni in developing a genotyping method. We examined genes in C. jejuni that exhibit binary status (present or absent between strains) within known plasticity regions, in order to identify a minimal subset of gene targets that provide high-resolution genetic fingerprints. Using CGH data from three studies as input, binary gene sets were identified with "Minimum SNPs" software. "Minimum SNPs" selects for the minimum number of targets required to obtain a predefined resolution, based on Simpson's index of diversity (D). After implementation of stringent criteria for gene presence/absence, eight binary genes were found that provided 100% resolution (D=1) of 20 C. jejuni strains. A real-time PCR assay was developed and tested on 181 C. jejuni and Campylobacter coli isolates, a subset of which have previously been characterized by multilocus sequence typing, flaA short variable region sequencing, and pulsed-field gel electrophoresis. In addition to the binary gene real-time PCR assay, we refined the seven-member single nucleotide polymorphism (SNP) real-time PCR assay previously described for C. jejuni and C. coli. By normalizing the SNP assay with the respective C. jejuni and C. coli ubiquitous genes, mapA and ceuE, the polymorphisms at each SNP could be determined without separate reactions for every polymorphism. We have developed and refined a rapid, highly discriminatory genotyping method for C. jejuni and C. coli that uses generic technology and is amenable to high-throughput analyses.

Animals↗

PubMed2006

Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria.

The expression and secretion signals of the Sep protein from Lactobacillus fermentum BR11 were used to direct export of two peptidoglycan hydrolases by Lb. fermentum BR11, Lactobacillus rhamnosus GG, Lactobacillus plantarum ATCC 14917 and Lactococcus lactis MG1363. The production levels, hydrolytic and bacteriocidal activities of the Listeria monocytogenes bacteriophage N-acetylmuramoyl-l-alanine amidase endolysin Ply511 and the glycylglycine endopeptidase lysostaphin were examined. Buffering of the growth media to a neutral pH allowed detection of Ply511 and lysostaphin peptidoglycan hydrolytic activity from all lactic acid bacteria. It was found that purified Ply511 has a pH activity range similar to that of lysostaphin with both enzymes functioning optimally under alkaline conditions. Supernatants from lactobacilli expressing lysostaphin reduced viability of methicillin resistant Staphylococcus aureus (MRSA) by approximately 8 log(10) CFU/ml compared to controls. However, supernatants containing Ply511 were unable to control L. monocytogenes growth. In coculture experiments, both Lb. plantarum and Lb. fermentum synthesizing lysostaphin were able to effectively reduce MRSA cell numbers by >7.4 and 1.7 log(10)CFU/ml, respectively, while lactic acid bacteria secreting Ply511 were unable to significantly inhibit the growth of L. monocytogenes. Our results demonstrate that lysostaphin and Ply511 can be expressed in an active form from different lactic acid bacteria and lysostaphin showed superior killing activity. Lactobacilli producing lysostaphin may have potential for in situ biopreservation in foodstuffs or for prevention of S. aureus infections.

Anti-Bacterial Agents↗

PubMed2006

Methicillin-resistant Staphylococcus aureus genotyping using a small set of polymorphisms.

The aim of this study was to identify a set of genetic polymorphisms that efficiently divides methicillin-resistant Staphylococcus aureus (MRSA) strains into groups consistent with the population structure. The rationale was that such polymorphisms could underpin rapid real-time PCR or low-density array-based methods for monitoring MRSA dissemination in a cost-effective manner. Previously, the authors devised a computerized method for identifying sets of single nucleotide polymorphisms (SNPs) with high resolving power that are defined by multilocus sequence typing (MLST) databases, and also developed a real-time PCR method for interrogating a seven-member SNP set for genotyping S. aureus. Here, it is shown that these seven SNPs efficiently resolve the major MRSA lineages and define 27 genotypes. The SNP-based genotypes are consistent with the MRSA population structure as defined by eBURST analysis. The capacity of binary markers to improve resolution was tested using 107 diverse MRSA isolates of Australian origin that encompass nine SNP-based genotypes. The addition of the virulence-associated genes cna, pvl and bbp/sdrE, and the integrated plasmids pT181, pI258 and pUB110, resolved the nine SNP-based genotypes into 21 combinatorial genotypes. Subtyping of the SCCmec locus revealed new SCCmec types and increased the number of combinatorial genotypes to 24. It was concluded that these polymorphisms provide a facile means of assigning MRSA isolates into well-recognized lineages.

Australia↗

PubMed2006

Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing.

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen Campylobacter jejuni. SNPs were identified in silico using the program 'Minimum SNPs', which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (D). The high-D SNPs identified in this study were derived from the combined C. jejuni/Campylobacter coli multilocus sequence typing (MLST) database. Seven SNPs were found that provided a D of 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high-D SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 C. jejuni isolates were subjected to MLST and flagellin A short variable region (flaA SVR) sequencing and combined with a population of 84 C. jejuni and C. coli isolates previously characterized by these methods. Within this collection of 153 isolates, 19 flaA SVR types (D=0.857) were identified, compared with 40 different STs (D=0.939). When MLST and flaA SVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided a D of 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of the flaA SVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/flaA SVR. This investigation has shown that a seven-member C. jejuni SNP typing assay, used in combination with sequencing of the flaA SVR, efficiently discriminates C. jejuni isolates.

Alleles↗

PubMed2006

Use of a single-nucleotide polymorphism genotyping system to demonstrate the unique epidemiology of methicillin-resistant Staphylococcus aureus in remote aboriginal communities.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has emerged as a major public health problem in Australia, as in many other parts of the world. High rates of CA-MRSA skin and soft tissue infection have been reported from Aboriginal communities. We used a single-nucleotide polymorphism (SNP) genotyping typing system based on the multilocus sequence type (MLST) database to investigate the epidemiology of CA-MRSA and methicillin-sensitive S. aureus (MSSA) over a 12-month period in three remote Aboriginal communities of Northern Australia. This was supplemented by real-time PCR for Panton-Valentine leukocidin (PVL) genes, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial susceptibility testing. S. aureus was recovered from pyoderma lesions on 221 occasions and throat swabs on 44 occasions. The median monthly recovery rate of S. aureus from skin sores was 58% (interquartile range, 62 to 78%), and there was no seasonal variation. Twenty-three percent of isolates were CA-MRSA; the proportion was similar across the communities and did not vary over the study period. Erythromycin resistance was found in 47% of CA-MRSA and 21% of MSSA. SNP-based typing identified 14 different clonal complexes (cc); however, cc75 was predominant, accounting for 71% of CA-MRSA isolates. These were confirmed as ST75-like by using an additional SNP and MLST of selected isolates. All but one of the cc75 isolates had SSCmec type IV (one had type V), and all were PVL negative. Monthly tracking of SNP-based cc types showed a highly dynamic process. ST75-MRSA-IV appears to be unique to the region and probably evolved de novo in remote Aboriginal communities.

Australia↗

PubMed2006

Staphylococcus aureus genotyping using novel real-time PCR formats.

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of "light upon extension" (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the "South West Pacific" and "Queensland" community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 "Aus-1/Aus-2" hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

Bacterial Typing Techniques↗

PubMed2005

BspA (CyuC) in Lactobacillus fermentum BR11 is a highly expressed high-affinity L-cystine-binding protein.

The BspA protein of Lactobacillus fermentum BR11 (BR11) is a cell envelope constituent that is similar to known solute-binding proteins and putative adhesins. BspA is required for L-cystine uptake and oxidative defense and is likely to be an L-cystine-binding protein. The aim of this study was to directly measure L-cystine-BspA binding and BspA expression. De-energized BR11 cells bound radiolabelled L-cystine with a Kd of 0.2 microM. A bspA mutant could not bind L-cystine. L-cystine-BR11 binding was unaffected by large excesses of L-glutamine, L-methionine, or collagen, indicating L-cystine specificity. BR11 and the bspA mutant were identical in their abilities to bind L-cysteine, indicating that L-cysteine is not a BspA ligand. BspA expression levels were deduced from radiolabelled L-cystine binding and it was found that there are 1-2 x 10(5) BspA molecules per cell, and that expression is slightly higher under oxidizing conditions. It is proposed that BspA be renamed CyuC.

Bacterial Proteins↗

PubMed2005

bla(SHV) Genes in Klebsiella pneumoniae: different allele distributions are associated with different promoters within individual isolates.

Extended-spectrum beta-lactamases (ESBLs) emerge by point mutation from non-extended-spectrum precursors. The aims of this study were to reveal the basis for variations in resistance levels found in a collection of 21 Klebsiella pneumoniae clinical isolates from Brisbane, Australia. Previous studies have shown that 20 of these isolates possess bla(SHV-11), bla(SHV-2a), and/or bla(SHV-12), and there is an association between the copy numbers of the ESBL-encoding genes and resistance levels. In this study, a real-time PCR method for interrogating the polymorphic sites at codons 238 and 240 was developed, and this confirmed the relationship between mutant gene copy numbers and resistance levels. The bla(SHV) promoter region was cloned from one of the ESBL-expressing isolates, and this showed that bla(SHV) genes exist downstream of two different promoters within this single isolate. These promoters have both been reported previously, and they differ by virtue of the presence or absence of an IS26 insertion. The bla(SHV) copy numbers in cis with the different promoters were measured, and the copy number of the IS26 promoter was correlated with resistance levels. Cloning and analysis of PCR products showed that different bla(SHV) variants existed in cis with individual promoters in individual isolates but that mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL-positive isolates without this promoter. It was concluded that bla(SHV) in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL-positive phenotype.

Alleles↗

PubMed2004

Identification, characterisation and specificity of a cell wall lytic enzyme from Lactobacillus fermentum BR11.

Screening of a genomic library with an antiserum raised against whole Lactobacillus fermentum BR11 cells identified a clone expressing an immunoreactive 37-kDa protein. Analysis of the 3010-bp DNA insert contained within the clone revealed four open reading frames (ORFs). One ORF encodes LysA, a 303 amino acid protein which has up to 35% identity with putative endolysins from prophages Lj928 and Lj965 from Lactobacillus johnsonii and Lp1 and Lp2 from Lactobacillus plantarum as well as with the endolysin of Lactobacillus gasseri bacteriophage Phiadh. The immunoreactive protein was shown to be encoded by a truncated ORF downstream of lysA which has similarity to glutamyl-tRNA synthetases. The N-terminus of LysA has sequence similarity with N-acetylmuramidase catalytic domains while the C-terminus has sequence similarity with putative cell envelope binding bacterial SH3b domains. C-terminal bacterial SH3b domains were identified in the majority of Lactobacillus bacteriophage endolysins. LysA was expressed in Escherichia coli and unusually was found to have a broad bacteriolytic activity range with activity against a number of different Lactobacillus species and against Lactococcus lactis, streptococci and Staphylococcus aureus. It was found that LysA is 2 and 8000 times more active against L. fermentum than L. lactis and Streptococcus pyogenes, respectively.

Amino Acid Sequence↗

PubMed2004

Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases.

A unified, bioinformatics-driven, single nucleotide polymorphism (SNP)-based approach to microbial genotyping has been developed. Multilocus sequence typing (MLST) databases consist of known variants of standardized housekeeping genes. Normally, seven fragments are defined; a sequence type (ST) consists of the variants of these fragments that are found in a particular isolate. A computer program that can identify highly informative sets of SNPs in entire MLST databases has been constructed. The SNPs either define a particular user-specified ST or provide a high value for Simpson's index of diversity (D), and may thus be generally applicable to that species. SNP sets that are diagnostic for Neisseria meningitidis ST-11 and ST-42, and high-D SNP sets for N. meningitidis and Staphylococcus aureus, were identified and real-time PCR methods to interrogate these SNPs were demonstrated. High-D SNP sets were also identified in other MLST databases. This widely applicable approach allows rapid genetic fingerprinting of infectious agents.

Algorithms↗

PubMed2004

Identification and characterization of the novel LysM domain-containing surface protein Sep from Lactobacillus fermentum BR11 and its use as a peptide fusion partner in Lactobacillus and Lactococcus.

Examination of supernatant fractions from broth cultures of Lactobacillus fermentum BR11 revealed the presence of a number of proteins, including a 27-kDa protein termed Sep. The amino-terminal sequence of Sep was determined, and the gene encoding it was cloned and sequenced. Sep is a 205-amino-acid protein and contains a 30-amino-acid secretion signal and has overall homology (between 39 and 92% identity) with similarly sized proteins of Lactobacillus reuteri, Enterococcus faecium, Streptococcus pneumoniae, Streptococcus agalactiae, and Lactobacillus plantarum. The carboxy-terminal 81 amino acids of Sep also have strong homology (86% identity) to the carboxy termini of the aggregation-promoting factor (APF) surface proteins of Lactobacillus gasseri and Lactobacillus johnsonii. The mature amino terminus of Sep contains a putative peptidoglycan-binding LysM domain, thereby making it distinct from APF proteins. We have identified a common motif within LysM domains that is shared with carbohydrate binding YG motifs which are found in streptococcal glucan-binding proteins and glucosyltransferases. Sep was investigated as a heterologous peptide expression vector in L. fermentum, Lactobacillus rhamnosus GG and Lactococcus lactis MG1363. Modified Sep containing an amino-terminal six-histidine epitope was found associated with the cells but was largely present in the supernatant in the L. fermentum, L. rhamnosus, and L. lactis hosts. Sep as well as the previously described surface protein BspA were used to express and secrete in L. fermentum or L. rhamnosus a fragment of human E-cadherin, which contains the receptor region for Listeria monocytogenes. This study demonstrates that Sep has potential for heterologous protein expression and export in lactic acid bacteria.

Amino Acid Sequence↗

PubMed2004

Genetic diversity among community methicillin-resistant Staphylococcus aureus strains causing outpatient infections in Australia.

Increasing reports of the appearance of novel nonmultiresistant methicillin-resistant Staphylococcus aureus MRSA (MRSA) strains in the community and of the spread of hospital MRSA strains into the community are cause for public health concern. We conducted two national surveys of unique isolates of S. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. A total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were MRSA. Approximately 54% of the MRSA isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. The majority of multiresistant MRSA isolates in both surveys belonged to two strains (strains AUS-2 and AUS-3), as determined by pulsed-field gel electrophoresis (PFGE) and resistogram typing. The 3 AUS-2 isolates and 10 of the 11 AUS-3 isolates selected for multilocus sequence typing (MLST) and staphylococcal chromosomal cassette mec (SCCmec) analysis were ST239-MRSA-III (where ST is the sequence type) and thus belonged to the same clone as the eastern Australian MRSA strain of the 1980s, which spread internationally. Four predominant clones of novel nonmultiresistant MRSA were identified by PFGE, MLST, and SCCmec analysis: ST22-MRSA-IV (strain EMRSA-15), ST1-MRSA-IV (strain WA-1), ST30-MRSA-IV (strain SWP), and ST93-MRSA-IV (strain Queensland). The last three clones are associated with community acquisition. A total of 14 STs were identified in the surveys, including six unique clones of novel nonmultiresistant MRSA, namely, STs 73, 93, 129, 75, and 80slv and a new ST. SCCmec types IV and V were present in diverse genetic backgrounds. These findings provide support for the acquisition of SCCmec by multiple lineages of S. aureus. They also confirm that both hospital and community strains of MRSA are now common in nonhospitalized patients throughout Australia.

Anti-Bacterial Agents↗

PubMed2004

mecA Locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone.

An emerging public health phenomenon is the increasing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections that are acquired outside of health care facilities. One lineage of community-acquired MRSA (CA-MRSA) is known as the Western Samoan phage pattern (WSPP) clone. The central aim of this study was to develop an efficient genotyping procedure for the identification of WSPP isolates. The approach taken was to make use of the highly variable region downstream of mecA in combination with a single nucleotide polymorphism (SNP) defined by the S. aureus multilocus sequence typing (MLST) database. The premise was that a combinatorial genotyping method that interrogated both a highly variable region and the genomic backbone would deliver a high degree of informative power relative to the number of genetic polymorphisms interrogated. Thirty-five MRSA isolates were used for this study, and their gene contents and order downstream of mecA were determined. The CA-MRSA isolates were found to contain a truncated mecA downstream region consisting of mecA-HVR-IS431 mec-dcs-Ins117, and a PCR-based method for identifying this structure was developed. The hospital-acquired isolates were found to contain eight different mecA downstream regions, three of which were novel. The Minimum SNPs computer software program was used to mine the S. aureus MLST database, and the arcC 272G polymorph was identified as 82% discriminatory for ST-30. A real-time PCR assay was developed to interrogate this SNP. We found that the assay for the truncated mecA downstream region in combination with the interrogation of arcC position 272 provided an unambiguous identification of WSPP isolates.

Bacterial Proteins↗

PubMed2003

Cystine uptake prevents production of hydrogen peroxide by Lactobacillus fermentum BR11.

BspA is an abundant surface protein from Lactobacillus fermentum BR11, and is required for normal cystine uptake. In previous studies, a mutant strain deficient in BspA (L. fermentum PNG201) was found to be sensitive to oxidative stress. In this study, the biochemical basis for this was explored. It was found that under aerobic batch culture conditions in de Mann-Rogosa-Sharpe medium, both L. fermentum BR11 and PNG201 entered stationary phase due to hydrogen peroxide accumulation. However, this took place at a lower optical density for PNG201 than for BR11. Measurements of hydrogen peroxide levels revealed that the BspA mutant strain overproduces this compound. Addition of 6 mM cystine to aerobic cultures was found to prevent hydrogen peroxide production by both the BR11 and PNG201 strains, but lower cystine concentrations depressed hydrogen peroxide production in BR11 more efficiently than in PNG201. Each mole of cystine was able to prevent the production of several moles of hydrogen peroxide by L. fermentum BR11, suggesting that hydrogen peroxide breakdown is dependent upon a thiol that cycles between reduced and oxidized states. It was concluded that peroxide breakdown by L. fermentum BR11 is dependent upon exogenous cystine. It is most probable that the imported L-cystine is catabolized by a cystathionine lyase and then converted into a thiol reductant for a peroxidase.

Bacterial Proteins↗

PubMed2003

Peptide surface display and secretion using two LPXTG-containing surface proteins from Lactobacillus fermentum BR11.

A locus encoding two repetitive proteins that have LPXTG cell wall anchoring signals from Lactobacillus fermentum BR11 has been identified by using an antiserum raised against whole L. fermentum BR11 cells. The first protein, Rlp, is similar to the Rib surface protein from Streptococcus agalactiae, while the other protein, Mlp, is similar to the mucus binding protein Mub from Lactobacillus reuteri. It was shown that multiple copies of mlp exist in the genome of L. fermentum BR11. Regions of Rlp, Mlp, and the previously characterized surface protein BspA were used to surface display or secrete heterologous peptides in L. fermentum. The peptides tested were 10 amino acids of the human cystic fibrosis transmembrane regulator protein and a six-histidine epitope (His(6)). The BspA promoter and secretion signal were used in combination with the Rlp cell wall sorting signal to express, export, and covalently anchor the heterologous peptides to the cell wall. Detection of the cell surface protein fusions revealed that Rlp was a significantly better surface display vector than BspA despite having lower cellular levels (0.7 mg per liter for the Rlp fusion compared with 4 mg per liter for the BspA fusion). The mlp promoter and encoded secretion signal were used to express and export large (328-kDa at 10 mg per liter) and small (27-kDa at 0.06 mg per liter) amino-terminal fragments of the Mlp protein fused to the His(6) and CFTR peptides or His(6) peptide, respectively. Therefore, these newly described proteins from L. fermentum BR11 have potential as protein production and targeting vectors.

Amino Acid Motifs↗

PubMed2002

Expression of a streptococcal glucosyltransferase as a fusion to a solute-binding protein in Lactobacillus fermentum BR11.

BspA is a non-covalently anchored cystine-binding protein from Lactobacillus fermentum BR11. It has previously been used to present antigens derived from infectious organisms on the L. fermentum BR11 cell surface. In this study, the capacity of BspA to present a very large polypeptide was tested. A temperature sensitive plasmid was constructed that encodes a 175-kDa chimeric protein consisting of a fusion between BspA and an N-terminally truncated derivative of the Streptococcus salivarius ATCC 25975 glucosyltransferase GtfJ. This plasmid was introduced into the L. fermentum genome. Integrants were able to incorporate 20-40 nmol sucrose derived glucose into glucan per ml culture per optical density unit. The glucosyltransferase activity was external to the cytoplasmic membrane and bound to the cell. Unlike native BspA, the BspA-GtfJ fusion could not be removed from the cell by 5 M LiCl wash.

Bacterial Proteins↗