Biomedical subjects

Louise M Hafner

Publications and source records attributed to Louise M Hafner.

4 recordsLinked to original sources

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 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

Structural changes in the cells of some bacteria during population growth: a Fourier transform infrared-attenuated total reflectance study.

Structural changes occurring in the cells of several bacteria during their growth curves have been investigated by Fourier transform infrared (FT-IR) spectroscopy using the sampling technique of attenuated total reflectance (ATR). Spectra reflect all of the components of the cells, including the cell walls, cell membranes, internal structures, and the cytoplasm. The bacteria studied were Bacillus stearothermophilus, Halobacterium salinarum, Halococcus morrhuae, and Acetobacter aceti. All species showed significant spectral changes during their growth curves, indicating structural changes in the cells during increases in cell numbers. The major change for B. stearothermophilus was in the lipid content, which was at a maximum during the exponential phase of the growth curve. For the halophiles H. salinarum and H. morrhuae, the major change was that the concentration of sulfate ion in the cells varied during the growth curve and was at a maximum during the mid-part of the exponential phase of the growth curve. A. aceti cells showed increasing polysaccharide content during the growth curve as well as maximum lipid content during the exponential phase of growth.

Acetobacter↗

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↗