New insights in the molecular biology and physiology of Streptococcus thermophilus revealed by comparative genomics

Streptococcus thermophilus is a major dairy starter used for the manufacture of yoghurt and cheese. The access to three genome sequences, comparative genomics and multilocus sequencing analyses suggests that this species recently emerged and is still undergoing a process of regressive evolution towards a specialised bacterium for growth in milk. Notably, S. thermophilus has maintained a well-developed nitrogen metabolism whereas its sugar catabolism has been subjected to a high level of degeneracy due to a paucity of carbon sources in milk. Furthermore, while pathogenic streptococci are recognised for a high capacity to expose proteins at their cell surface in order to achieve cell adhesion or to escape the host immune system, S. thermophilus has nearly lost this unique feature as well as many virulence-related functions. Although gene decay is obvious in S. thermophilus genome evolution, numerous small genomic islands, which were probably acquired by horizontal gene transfer, comprise important industrial phenotypic traits such as polysaccharide biosynthesis, bacteriocin production, restriction-modification systems or oxygen tolerance.     

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

It has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli . Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD  ::Tn 5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD -mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA , recF , recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD ⁺ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.     

Proteome phenotyping of acid stress-resistant mutants of Lactococcus lactis MG1363

To cope with medium acidity, Lactococcus lactis has evolved a number of inducible mechanisms commonly referred as acid stress response. To better understand the molecular basis of this response, several mutants constitutively tolerant to acidity were previously obtained by insertional random mutagenesis of L. lactis MG1363. Mutants in which the GMP synthase gene (i.e. guaA), the (p)ppGpp synthase gene (i.e. relA*) or the high affinity phosphate transport system (i.e. pstS) are inactivated are further characterized in this study. 2-DE was performed and showed that 42, 26, and 35 protein spots are positively deregulated in the guaA, relA*, and pstS mutants, respectively, as compared to the wild-type strain. Most of these proteins were identified by MS. Proteomes comparison of the mutants guaA, relA*, and pstS as well as the acid adaptation proteome of the wild-type strain revealed (i) the presence of numerous overlaps and (ii) that only five proteins were overexpressed in the four conditions, suggesting that these proteins play a crucial role in the constitutive acid stress tolerance of the mutants and in the acid tolerance response of the wild-type strain.     

Systematic localisation of proteins fused to the green fluorescent protein in Bacillus subtilis: identification of new proteins at the DNA replication factory

Construction and microscopic imaging of protein fusions to green fluorescent protein (GFP) have revolutionised our understanding of bacterial structure and function. We have undertaken a systematic study of the localisation of over 100 Bacillus subtilis proteins, following the development of high-throughput construction and analysis procedures. We focused on proteins linked in various ways to the DNA replication machinery, as well as on proteins exemplifying a range of other cellular functions and structures. The results validate the approach as a way of obtaining systematic protein localisation information. They also provide a range of novel biological insights, particularly through the identification of a number of proteins not previously known to be associated with the DNA replication factory.     

Recent genetic transfer between Lactococcus lactis and enterobacteria

The genome sequence of Lactococcus lactis revealed that the ycdB gene was recently exchanged between lactococci and enterobacteria. The present study of ycdB orthologs suggests that L. lactis was probably the gene donor and reveals three instances of gene transfer to enterobacteria. Analysis of ycdB gene transfer between two L. lactis subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris, indicates that the gene can be mobilized, possibly by conjugation.     

Targeted disruption of the PDZK1 gene by homologous recombination

Proteins containing PDZ domains are involved in a large number of biological functions, including protein scaffolding, organization of ion channels, and signal transduction. We recently identified a novel PDZ domain-containing protein, PDZK1, that is selectively expressed in normal tissues, where it is associated and colocalized with MAP17, a small 17-kDa membrane-associated protein; cMOAT, an organic anion transporter implicated in multidrug resistance; and the type IIa Na/Pi cotransporter. The protein cluster formed by PDZK1, MAP17, and cMOAT is upregulated in a significant number of human carcinomas originating in the colon, breast, lung, and kidney. In order to better define the function of PDZK1 in the protein cluster and its potential role in the organization of ion channels, we generated a PDZK1 knockout mouse. While PDZK1-deficient mice developed normally, did not display any gross phenotypic abnormalities, and were fecund, lack of PDZK1 resulted in modulation of expression of selective ion channels in the kidney, as well as increased serum cholesterol levels. However, no significant redistribution of proteins known to interact with PDZK1, such as MAP17, cMOAT, and the type IIa Na/Pi cotransporter, was observed. The absence of a more significant phenotype in PDZK1-deficient mice may be due to functional compensation by other PDZ domain-containing proteins, which could be instrumental in determining the location of interacting proteins such as ion channels and other membrane-associated proteins in defined areas of the plasma membrane.