Biomedical subjects

Mário A Santos

Publications and source records attributed to Mário A Santos.

5 recordsLinked to original sources

PubMed2006

The minor capsid protein gp7 of bacteriophage SPP1 is required for efficient infection of Bacillus subtilis.

Gp7 is a minor capsid protein of the Bacillus subtilis bacteriophage SPP1. Homologous proteins are found in numerous phages but their function remained unknown. Deletion of gene 7 from the SPP1 genome yielded a mutant phage (SPP1del7) with reduced burst-size. SPP1del7 infections led to normal assembly of virus particles whose morphology, DNA and protein composition was undistinguishable from wild-type virions. However, only approximately 25% of the viral particles that lack gp7 were infectious. SPP1del7 particles caused a reduced depolarization of the B. subtilis membrane in infection assays suggesting a defect in virus genome traffic to the host cell. A higher number of SPP1del7 DNA ejection events led to abortive release of DNA to the culture medium when compared with wild-type infections. DNA ejection in vitro showed that no detectable gp7 is co-ejected with the SPP1 genome and that its presence in the virion correlated with anchoring of released DNA to the phage particle. The release of DNA from wild-type phages was slower than that from SPP1del7 suggesting that gp7 controls DNA exit from the virion. This feature is proposed to play a central role in supporting correct routing of the phage genome from the virion to the cell cytoplasm.

Bacillus Phages↗

PubMed2006

Leuconostoc pseudoficulneum sp. nov., isolated from a ripe fig.

Six strains of lactic acid bacteria (LAB) were isolated from a ripe fig. These strains constituted a highly homogeneous, but distinct, cluster that was separate from other LAB species in a polyphasic approach including dot-blot DNA-DNA hybridization, SDS-PAGE whole-cell protein profiling, carbohydrate fermentation ability, growth characteristics, enzymic profiling, pulsed-field gel electrophoresis macrorestriction analysis and RFLPs. Phylogenetic analysis based on 16S rRNA gene sequencing positioned a representative strain, LC51(T), in a distinct line of descent within the recently described clade comprising Leuconostoc ficulneum, Leuconostoc fructosum and Leuconostoc durionis; L. ficulneum was its closest neighbour (98 % sequence similarity). DNA-DNA hybridization values and chemotaxonomic and biochemical characteristics, including enzymic profiles detected with API ZYM microtubes, confirmed that this group of strains is distinct from L. ficulneum and represents a novel species within the genus Leuconostoc. Taking into account the common origin and phylogenetic proximity, the name Leuconostoc pseudoficulneum sp. nov. is proposed. Strain LC51(T) (=DSM 15468(T) = CECT 5759(T)) is the type strain; the DNA G + C content of this strain is 44.5 mol%.

Base Composition↗

PubMed2004

The high-resolution functional map of bacteriophage SPP1 portal protein.

An essential component in the assembly of nucleocapsids of tailed bacteriophages and of herpes viruses is the portal protein that is located at the unique vertex of the icosahedral capsid through which DNA movements occur. A library of mutations in the bacteriophage SPP1 portal protein (gp6) was generated by random mutagenesis of gene 6. Screening of the library allowed identification of 67 single amino acid substitutions that impair portal protein function. Most of the mutations cluster within stretches of a few amino acids in the gp6 carboxyl-terminus. The mutations were divided into five classes according to the step of virus assembly that they impair: (1) production of stable gp6; (2) interaction of gp6 with the minor capsid protein gp7; (3) incorporation of gp6 in the procapsid structure; (4) DNA packaging; and (5) sizing of the packaged DNA molecule. Most of the mutations fell in classes 3 and 4. This is the first high-resolution functional map of a portal protein, in which its function at different steps of viral assembly can be directly correlated with specific regions of its sequence. The work provides a framework for the understanding of central processes in the assembly of viruses that use specialized portals to govern entry and exit of DNA from the viral capsid.

Amino Acid Substitution↗

PubMed2004

Bacillus subtilis operon encoding a membrane receptor for bacteriophage SPP1.

The results reported here have identified yueB as the essential gene involved in irreversible binding of bacteriophage SPP1 to Bacillus subtilis. First, a deletion in an SPP1-resistant (pha-2) strain, covering most of the yueB gene, could be complemented by a xylose-inducible copy of yueB inserted at amyE. Second, disruption of yueB by insertion of a pMutin4 derivative resulted in a phage resistance phenotype regardless of the presence or absence of IPTG (isopropyl-beta-D-thiogalactopyranoside). YueB homologues are widely distributed in gram-positive bacteria. The protein Pip, which also serves as a phage receptor in Lactococcus lactis, belongs to the same family. yueB encodes a membrane protein of approximately 120 kDa, detected in immunoblots together with smaller forms that may be processed products arising from cleavage of its long extracellular domain. Insertional inactivation of yueB and the surrounding genes indicated that yueB is part of an operon which includes at least the upstream genes yukE, yukD, yukC, and yukBA. Disruption of each of the genes in the operon allowed efficient irreversible adsorption, provided that yueB expression was retained. Under these conditions, however, smaller plaques were produced, a phenotype which was particularly noticeable in yukE mutant strains. Interestingly, such reduction in plaque size was not correlated with a decreased adsorption rate. Overall, these results provide the first demonstration of a membrane-bound protein acting as a phage receptor in B. subtilis and suggest an additional involvement of the yukE operon in a step subsequent to irreversible adsorption.

Amino Acid Sequence↗

PubMed1998

Physical map of the genome of Oenococcus oeni PSU-1 and localization of genetic markers.

A physical map of the chromosome of Oenococcus oeni PSU-1 was constructed. This represents the first map for a strain of this species. A total of 37 restriction sites for the rare-cutting endonucleases Ascl, Fsel, Notl and Sfil were mapped on the chromosome, which was found to be circular with an estimated size of 1857 kb. Fragment order was determined using several approaches: analysis of partial and double digestions, two-dimensional pulsed-field gel electrophoresis, isolation of linking clones, and Southern hybridization with labelled restriction fragments both from PSU-1 and from O. oeni strain GM. Oenococcal genes alsS/alsD, mleA and mir, two phage attachment sites and recurrent sequences such as IS1165-like elements and rrn loci were located on the physical map. Specific fragments hybridizing with gene probes from Lactococcus lactis, Leuconostoc mesenteroides and Bacillus subtilis were also identified. The two ribosomal operons have been precisely located and their transcription direction determined.

Bacteriophages↗