The ami locus of the Gram-positive bacterium Streptococcus pneumoniae is similar to binding protein-dependent transport operons of Gram-negative bacteria

The complete nucleotide sequence of the ami locus of Streptococcus pneumoniae revealed the presence of six open reading frames, amiABCDEF. The predicted Ami proteins are probably involved in a transport system. The AmiA, C, D, E, and F proteins exhibit homology with components of the oligopeptide permeases (opp) of Salmonella typhimurium and Escherichia coli. Intriguingly, the AmiB protein is homologous to ArsC, a cytosolic modifier subunit of the anion pump encoded by the arsenical resistance operon of the R-factor R773 from E. coli. Data are presented which indicate that Ami is indeed a transport system.     

Strand targeting signal(s) for in vivo mutation avoidance by post-replication mismatch repair in Escherichia coli

Summary The involvement of GATC sites in directing mismatch correction for the elimination of replication errors in Escherichia coli was investigated in vivo by analyzing mutation rates for a gene carried on a series of related plasmids that contain 2, 1 and 0 such sites. This gene encoding chloramphenicol acetyl transferase (Cat protein) was inactivated by a point mutation. In vivo mutations restoring resistance to chloramphenicol were scored in mismatch repair proficient (mut ⁺) and deficient (mutHLS^-) strains. In mut ⁺ cells, reduction of GATC sites from 2 to 0 increased mutation rates approximately 10-fold. Removal of the GATC site distal to the cat ^- mutation increased the rate of mutation less than 2-fold, indicating that mismatch repair can proceed normally with a single site. The mutation rate increased 3-fold after removal of the GATC site proximal to the mutation. In the absence of a GATC site, mutL^- and mutS^- strains exhibited a 2- to 3-fold increased mutation rate as compared to isogenic mutH^- and mut ⁺ strains. This indicates that 50%–70% of replication errors can be corrected in a mutLS-dependent way in the absence of any GATC site to target mismatch correction to newly synthesized DNA strands. Other strand targeting signals, possibly single strand discontinuities, might be used in mutLS-dependent repair     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

Competence pheromone, oligopeptide permease, and induction of competence in Streptococcus pneumoniae

An unmodified heptadecapeptide pheromone capable of eliciting competence for genetic transformation in Streptococcus pneumoniae has recently been identified and characterized. In considering possible signaltransduction mechanisms for the peptide, the previously characterized Ami oligopeptide permease and the three highly homologous oligopeptide-binding lipoproteins, AmiA. AliA, and AliB, appeared to be good candidates for receptors. We therefore compared the spontaneous transformability of Ami, AliA and AliB mutants to that of an isogenic wild-type strain and we investigated the response of the various mutants to treatment with synthetic competence-stimulating peptide (CSP). Our results clearly demonstrate that neither Ami nor any of the three highly homologous oligopeptide-binding lipoproteins identified so far in S. pneumoniae are required for competence induction following treatment with synthetic CSP. Although the existence of a fourth unidentified oligopeptide-binding lipoprotein and/or a second oligopeptide permease operon could not be completely ruled out, we favour the hypothesis that CSP signal transmission rather involves a two-component regulatory system. Although none of the single or double Ami and Ali mutants tested appeared severely affected for competence, an exceptional aliB plasmid-insertion mutation abolished competence completely. In addition, the triple AmiA-AliA-AliB mutant differed from wild type in showing no sharp peak of competence but exhibiting transformability throughout the exponential phase of growth. These and previous observations are discussed and a general hypothesis is proposed to account for the modulation of competence by peptide permease mutants in S. pneumoniae.     

Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation.

The efficiency of transformation of point mutations depends upon base pair mismatches during the recombination process. For low-efficiency markers, the genetic information carried on the donor deoxyribonucleic acid is preferentially lost. To understand this elimination process, we investigated the effect of the size of donor deoxyribonucleic acid on the relative efficiency of low-efficiency point mutations. The deoxyribonucleic acid was shortened either by mechanical shearing or by restriction enzyme treatments. The results indicate that transformation by low-efficiency markers was not affected by shortening the distance between them and the end of the molecule any more than was transformation by the other markers. Moreover, no lethal event could be detected for either cell or chromosomal marker survival. These data do not exclude the double-strand-break hypothesis that was proposed to explain the loss of genetic information for low-efficiency markers, but they offer no support for it.     

A Type IV Pilus Mediates DNA Binding during Natural Transformation in Streptococcus pneumoniae

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.