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Background: Gut microbial gene richness and specific bacterial species are associated with metabolic risk markers in humans, but the impact of host physiology and dietary habits on the link between the gut microbiota and metabolic markers remain unclear. The objective of this study was to identify gut metagenomic markers associated with estimates of insulin resistance, lipid metabolism and inflammation in obesity, and to explore whether the associations between metagenomic and metabolic markers persisted after adjustment for body fat, age and habitual dietary intake., Methods: Faecal DNA from 53 women with obesity was analysed through quantitative metagenomic sequencing and analysis, and a systematic search was performed for bacterial genes associated with estimates of insulin resistance, inflammation and lipid metabolism. Subsequently, the correlations between metagenomic species and metabolic markers were tested by linear regression models, with and without covariate adjustment., Results: One hundred and fourteen metagenomic species correlated with metabolic markers (P<0.001) including Akkermansia muciniphila, Bilophila wadsworthia, Bifidobacterium longum and Faecalibacterium prausnitzii, but also species not previously associated with metabolic markers including Bacteroides faecis and Dorea longicatena. The majority of the identified correlations between bacterial species and metabolic markers persisted after adjustment for differences in body fat, age and dietary macronutrient composition; however, the negative correlation with insulin resistance observed for B. longum and F. prausnitzii appeared to be modified by the intake of dietary fibre and fat, respectively., Conclusions: This study shows that several gut bacterial species are linked to metabolic risk markers in obesity, also after adjustment for potential confounders, such as long-term diet composition. The study supports the use of gut metagenomic markers for metabolic disease prediction and warrants further investigation of causality.

Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification.

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) is a representative of the group of lactic acid-producing bacteria, mainly known for its worldwide application in yogurt production. The genome sequence of this bacterium has been determined and shows the signs of ongoing specialization, with a substantial number of pseudogenes and incomplete metabolic pathways and relatively few regulatory functions. Several unique features of the L. bulgaricus genome support the hypothesis that the genome is in a phase of rapid evolution. (i) Exceptionally high numbers of rRNA and tRNA genes with regard to genome size may indicate that the L. bulgaricus genome has known a recent phase of important size reduction, in agreement with the observed high frequency of gene inactivation and elimination; (ii) a much higher GC content at codon position 3 than expected on the basis of the overall GC content suggests that the composition of the genome is evolving toward a higher GC content; and (iii) the presence of a 47.5-kbp inverted repeat in the replication termination region, an extremely rare feature in bacterial genomes, may be interpreted as a transient stage in genome evolution. The results indicate the adaptation of L. bulgaricus from a plant-associated habitat to the stable protein and lactose-rich milk environment through the loss of superfluous functions and protocooperation with Streptococcus thermophilus.

The regulation of initiation of DNA replication is crucial to ensure that the genome is replicated only once per cell cycle. In the Gram-positive bacterium Bacillus subtilis, the function of the YabA protein in initiation control was assigned based on its interaction with the DnaA initiator and the DnaN sliding clamp in the yeast two-hybrid and on the overinitiation phenotype observed in a yabA null strain. However, YabA is unrelated to known regulators of initiation and interacts with several additional proteins that could also be involved directly or not in initiation control. Here, we investigated the specific role of YabA interactions with DnaA and DnaN in initiation control by identifying single amino acid changes in YabA that disrupted solely the interaction with DnaA or DnaN. These disruptive mutations delineated specific interacting surfaces involving a Zn2+-cluster structure in YabA. In B. subtilis, these YabA interaction mutations abolished both initiation control and the formation of YabA foci at the replication factory. Upon coexpression of deficient YabA mutants, mixed oligomers formed foci at the replisome and restored initiation control, indicating that YabA acts within a heterocomplex with DnaA and DnaN. In agreement, purified YabA oligomerized and formed complexes with DnaA and DnaN. These findings underscore the functional association of YabA with the replication machinery, indicating that YabA regulates initiation through coupling with the elongation of replication.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.

Primosomes are nucleoprotein assemblies designed for the activation of DNA replication forks. Their primary role is to recruit the replicative helicase onto single-stranded DNA. The "replication restart" primosome, defined in Escherichia coli, is involved in the reactivation of arrested replication forks. Binding of the PriA protein to forked DNA triggers its assembly. PriA is conserved in bacteria, but its primosomal partners are not. In Bacillus subtilis, genetic analysis has revealed three primosomal proteins, DnaB, DnaD, and DnaI, that have no obvious homologues in E. coli. Interestingly, they are involved in primosome function both at arrested replication forks and at the chromosomal origin. Our biochemical analysis of the DnaB and DnaD proteins unravels their role in primosome assembly. They are both multimeric and bind individually to DNA. Furthermore, DnaD stimulates DnaB binding activities. DnaD alone and the DnaD/DnaB pair interact specifically with PriA of B. subtilis on several DNA substrates. This suggests that the nucleoprotein assembly is sequential in the PriA, DnaD, DnaB order. The preferred DNA substrate mimics an arrested DNA replication fork with unreplicated lagging strand, structurally identical to a product of recombinational repair of a stalled replication fork.

DNA replication in bacteria is carried out by a multiprotein complex, which is thought to contain only one essential DNA polymerase, specified by the dnaE gene in Escherichia coli and the polC gene in Bacillus subtilis. Bacillus subtilis genome analysis has revealed another DNA polymerase gene, dnaE(BS), which is homologous to dnaE. We show that, in B. subtilis, dnaE(BS) is essential for cell viability and for the elongation step of DNA replication, as is polC, and we conclude that there are two different essential DNA polymerases at the replication fork of B. subtilis, as was previously observed in eukaryotes. dnaE(BS) appears to be involved in the synthesis of the lagging DNA strand and to be associated with the replication factory, which suggests that two different polymerases carry out synthesis of the two DNA strands in B. subtilis and in many other bacteria that contain both polC and dnaE genes.

Phenotypes of Bacillus subtilis priA mutants suggest that they are deficient in the restart of stalled chromosomal replication forks. The presumed activity of PriA in the restart process is to promote the assembly of a multiprotein complex, the primosome, which functions to recruit the replication fork helicase onto the DNA. We have proposed previously that three proteins involved in the initiation of replication at oriC in B. subtilis, DnaB, DnaD and DnaI, are components of the PriA primosome in this bacterium. However, the involvement of these proteins in replication restart has not yet been studied. Here, we describe dnaB mutations that suppress the phenotypes of B. subtilis priA mutants. In a representative mutant, the DnaC helicase is loaded onto single-stranded DNA in a PriA-independent, DnaD- and DnaI-dependent manner. These observations confirm that DnaB, DnaD and DnaI are primosomal proteins in B. subtilis. Moreover, their involvement in the suppression of priA phenotypes shows that they participate in replication fork restart in B. subtilis.

Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base., (Copyright 2001 Academic Press.)

Aims: The detection of growth-inhibiting factors produced by Lactobacillus delbrueckii., Methods and Results: A bioscreen assay was developed to study the effect of Lact. delbrueckii culture supernatant fluids on the growth of phylogenically or functionally related bacteria in broth cultures. Several growth-inhibiting factors could be distinguished based on differential effects on different test strains, separation by ultrafiltration and sensitivity to heat, proteinase treatment or catalase addition., Conclusion: Lactobacillus delbrueckii strain VI1007 was found to produce at least three growth-inhibiting factors, other than lactic acid, when grown under microaerobic conditions in MRS broth. These included H2O2 and a bacteriocin-like, heat- and proteinase-sensitive bactericidal molecule or complex with a molecular weight greater than 50 kDa. A third factor inhibited the growth of Streptococcus thermophilus., Significance and Impact of the Study: The assay system used allows the detection of subtle interactions between strains, that are likely to be of ecological importance in mixed cultures but would go unnoticed in classical agar diffusion tests.

The transcription of 16 genes encoding 12 peptidases (pepC, pepN, pepX, pepP, pepA, pepF2, pepDA1, pepDA2, pepQ, pepT, pepM, and pepO1), P(I) and P(III) proteinases (prtP1 and prtP3), and three transport systems (dtpT, dtpP, and opp-pepO1) of Lactococcus lactis MG1363 was analyzed in response to different environmental factors. Promoter fusions with luciferase reporter genes and/or mRNA analysis were used to study the effects of sugar sources, growth at 37 degrees C, and peptide supply on the transcription of these genes. Only transcription of the pepP gene is modulated by the source of sugar. The presence of potential catabolite-responsive element (CRE) boxes in its promoter region suggests that expression of this gene is directly controlled by catabolic repression. Elevated temperature had no significant effect on the level of transcription of these genes. prtP1, prtP3, pepC, pepN, pepX, and the opp-pepO1 operon are the most highly expressed genes in chemically defined medium, and their expression is repressed 5- to 150-fold by addition of peptide sources such as Casitone in the medium. Moreover, the transcription of prtP1, prtP3, pepC, pepN, and the opp-pepO1 operon is repressed two- to eight-fold by the dipeptides leucylproline and prolylleucine. The transcription of pepDA2 might also be repressed by the peptide sources, but this effect is not observed on the regulation of dtpT, pepP, pepA, pepF2, pepDA1, pepQ, pepT, pepM, and the dtpP operon. The significance of these results with respect to the functions of different components of the proteolytic system in L. lactis are discussed.

Proteolysis is essential for supplying Lactococcus lactis with amino acids during growth in milk. Expression of the major components of the L. lactis proteolytic system, including the cell wall proteinase (PrtP), the oligopeptide transport system (Opp) and at least four intracellular peptidases (PepO1, PepN, PepC, PepDA2), was shown previously to be controlled negatively by a rich nitrogen source. The transcription of prtP, opp-pepO1, pepN and pepC genes is regulated by dipeptides in the medium. Random insertion mutants derepressed for nitrogen control in the expression of the oligopeptide transport system were isolated using an opp-lacZ fusion. A third of the mutants were targeted in the same locus. The product of the inactivated gene shared 48% identity with CodY from Bacillus subtilis, a pleiotropic repressor of the dipeptide permease operon (dpp) and several genes including genes involved in amino acid degradation and competence induction. The signal controlling CodY-dependent repression was searched for by analysing the response of the opp-lux fusion to the addition of 67 dipeptides with different amino acid compositions. Full correlation was found between the dipeptide content in branched-chain amino acids (BCAA; isoleucine, leucine or valine) and their ability to mediate the repression of opp-pepO1 expression. The repressive effect resulting from specific regulatory dipeptides was abolished in L. lactis mutants affected in terms of their transport or degradation into amino acids, showing that the signal was dependent on the BCAA pool in the cell. Lastly, the repression of opp-pepO1 expression was stronger in a mutant unable to degrade BCAAs, underlining the central role of BCAAs as a signal for CodY activity. This pattern of regulation suggests that, in L. lactis and possibly other Gram-positive bacteria, CodY is a pleiotropic repressor sensing nutritional supply as a function of the BCAA pool in the cell.

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.

Lactococcus lactis is a nonpathogenic AT-rich gram-positive bacterium closely related to the genus Streptococcus and is the most commonly used cheese starter. It is also the best-characterized lactic acid bacterium. We sequenced the genome of the laboratory strain IL1403, using a novel two-step strategy that comprises diagnostic sequencing of the entire genome and a shotgun polishing step. The genome contains 2,365,589 base pairs and encodes 2310 proteins, including 293 protein-coding genes belonging to six prophages and 43 insertion sequence (IS) elements. Nonrandom distribution of IS elements indicates that the chromosome of the sequenced strain may be a product of recent recombination between two closely related genomes. A complete set of late competence genes is present, indicating the ability of L. lactis to undergo DNA transformation. Genomic sequence revealed new possibilities for fermentation pathways and for aerobic respiration. It also indicated a horizontal transfer of genetic information from Lactococcus to gram-negative enteric bacteria of Salmonella-Escherichia group.

Replication arrests due to the lack or the inhibition of replicative helicases are processed by recombination proteins. Consequently, cells deficient in the Rep helicase, in which replication pauses are frequent, require the RecBCD recombination complex for growth. rep recA mutants are viable and display no growth defect at 37 or 42 degrees C. The putative role of chaperone proteins in rep and rep recA mutants was investigated by testing the effects of dnaK mutations. dnaK756 and dnaK306 mutations, which allow growth of otherwise wild-type Escherichia coli cells at 40 degrees C, are lethal in rep recA mutants at this temperature. Furthermore, they affect the growth of rep mutants, and to a lesser extent, that of recA mutants. We conclude that both rep and recA mutants require DnaK for optimal growth, leading to low viability of the triple (rep recA dnaK) mutant. rep recA mutant cells form colonies at low efficiency when grown to exponential phase at 30 degrees C. Although the plating defect is not observed at a high temperature, it is not suppressed by overexpression of heat shock proteins at 30 degrees C. The plating defect of rep recA mutant cells is suppressed by the presence of catalase in the plates. The cryosensitivity of rep recA mutants therefore results from an increased sensitivity to oxidative damage upon propagation at low temperatures.

The RepE protein of the broad host range pAMbeta1 plasmid from Gram-positive bacteria is absolutely required for replication. To elucidate its role, we purified the protein to near homogeneity and analyzed its interactions with different nucleic acids using gel retardation assays and footprinting experiments. We show that RepE is monomeric in solution and binds specifically, rapidly, and durably to the origin at a unique double-stranded binding site immediately upstream from the initiation site of DNA replication. The binding induces only a weak bend (31 degrees ). Unexpectedly, RepE also binds nonspecifically to single-stranded DNA with a 2-4-fold greater affinity than for double-stranded origin. On a supercoiled plasmid, RepE binding to the double-stranded origin leads to the denaturation of the AT-rich sequence immediately downstream from the binding site to form an open complex. This open complex is atypical since (i) its formation requires neither multiple RepE binding sites on the double-stranded origin nor strong bending of the origin, (ii) it occurs in the absence of any cofactors (only RepE and supercoiling are required), and (iii) its melted region serves as a substrate for RepE binding. These original properties together with the fact that pAMbeta1 replication depends on a transcription step through the origin on DNA polymerase I to initiate replication and on a primosome to load the replisome suggest that the main function of RepE is to assist primer generation at the origin.

We have studied DNA recombination between 513 bp tandem direct repeats present in a kanamycin resistance gene inserted in the Bacillus subtilis chromosome. Tandem repeat deletion was not significantly affected by a recA mutation. However, recombination was stimulated by mutations in genes encoding replication proteins, including the primosomal proteins DnaB, DnaD and the DnaG primase, the putative DNA polymerase III subunits PolC, DnaN and DnaX, as well as the DNA polymerase DnaE. Hyper-recombination was found to be dependent on RecA in the dnaE, dnaN and dnaX mutants, whereas the dnaG and dnaD mutants stimulated recombination independently of RecA. Altogether, these data show that both RecA-dependent and RecA-independent mechanisms contribute to recombination between tandem repeats in B. subtilis and that both types of recombination are stimulated by replication mutations.

We report the genetic organisation of six prophages present in the genome of Lactococcus lactis IL1403. The three larger prophages (36-42 kb), belong to the already described P335 group of temperate phages, whereas the three smaller ones (13-15 kb) are most probably satellites relying on helper phage(s) for multiplication. These data give a new insight into the genetic structure of lactococcal phage populations. P335 temperate phages have variable genomes, sharing homology over only 10-33% of their length. In contrast, virulent phages have highly similar genomes sharing homology over >90% of their length. Further analysis of genetic structure in all known groups of phages active on other bacterial hosts such as Escherichia coli, Bacillus subtilis, MYCOBACTERIUM: and Streptococcus thermophilus confirmed the existence of two types of genetic structure related to the phage way of life. This might reflect different intensities of horizontal DNA exchange: low among purely virulent phages and high among temperate phages and their lytic homologues. We suggest that the constraints on genetic exchange among purely virulent phages reflect their optimal genetic organisation, adapted to a more specialised and extreme form of parasitism than temperate/lytic phages.

The holD gene codes for the psi subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the gamma complex clamp loader. A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions. In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA. They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant. The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature. DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD. We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype.

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h(-1). MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.

DNA ligases are grouped into two families, ATP-dependent and NAD-dependent, according to the cofactor required for their activity. A surprising capability of both kinds of ligases to complement for one another in vivo has been observed. Bacillus subtilis harbours one NAD-dependent ligase, YerG, and two ATP-dependent ligases, YkoU and YoqV, this last one being encoded by the 134 kb lysogenic bacteriophage SPss and consisting of a single adenylation domain typical of ATP-dependent ligases. Because the genetics of ligases in B.subtilis had not been studied previously, the genes encoding for one ligase of each kind, yerG and yoqV, were investigated. We found that the yerG gene was essential in B.subtilis. This suggests that none of the ATP-dependent ligases was able to complement the yerG defect. In addition, the ATP-dependent ligase encoded by yoqV, when cloned on a plasmid under appropriate expression signals, was unable to rescue a yerG mutant strain. The two B.subtilis ligase genes yerG and yoqV were also introduced in an Escherichia coli strain encoding a thermosensitive ligase (ligts), and whereas yoqV did not complement the ligts defects, yerG fully complemented the growth and UV sensitivity defects of the lig mutant. We propose to rename the yerG and yoqV genes of B.subtilis ligA and ligB respectively.

Replication fork arrest can cause DNA double-strand breaks (DSBs). These DSBs are caused by the action of the Holliday junction resolvase RuvABC, indicating that they are made by resolution of Holliday junctions formed at blocked forks. In this work, we study the homologous recombination functions required for RuvABC-mediated breakage in cells deficient for the accessory replicative helicase Rep or deficient for the main Escherichia coli replicative helicase DnaB. We show that, in the rep mutant, RuvABC-mediated breakage occurs in the absence of the homologous recombination protein RecA. In contrast, in dnaBts mutants, most of the RuvABC-mediated breakage depends on the presence of RecA, which suggests that RecA participates in the formation of Holliday junctions at forks blocked by the inactivation of DnaB. This action of RecA does not involve the induction of the SOS response and does not require any of the recombination proteins essential for the presynaptic step of homologous recombination, RecBCD, RecF or RecO. Consequently, our observations suggest a new function for RecA at blocked replication forks, and we propose that RecA acts by promoting homologous recombination without the assistance of known presynaptic proteins.

We describe a method to generate in vivo collections of mutants orders of magnitude larger than previously possible. The method favors accumulation of mutations in the target gene, rather than in the host chromosome. This is achieved by propagating the target gene on a plasmid, in Escherichia coli cells, within the region preferentially replicated by DNA polymerase I (Pol I), which replicates only a minor fraction of the chromosome. Mutagenesis is enhanced by a conjunction of a Pol I variant that has a low replication fidelity and the absence of the mutHLS system that corrects replication errors. The method was tested with two reporter genes, encoding lactose repressor or lipase. The proportion of mutants in the collection was estimated to reach 1% after one cycle of growth and 10% upon prolonged cell cultivation, resulting in collections of 10(12)-10(13) mutants per liter of cell culture. The extended cultivation did not affect growth properties of the cells. We suggest that our method is well suited for generating protein variants too rare to be present in the collections established by methods used previously and for isolating the genes that encode such variants by submitting the cells of the collections to appropriate selection protocols.

The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six ytr genes form a single operon, transcribed from a putative sigma(A)-dependent promoter present upstream of ytrA. YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation. It is known that B. subtilis produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation. When either the entire ytr operon or its last gene, ytrF, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient. We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation.

The alpha-acetolactate decarboxylase (ALDC) gene, aldB, is the penultimate gene of the leu-ilv-ald operon, which encodes the three branched-chain amino acid (BCAA) biosynthesis genes in Lactococcus lactis. Its product plays a dual role in the cell: (i) it catalyzes the second step of the acetoin pathway, and (ii) it controls the pool of alpha-acetolactate during leucine and valine synthesis. It can be transcribed from the two promoters present upstream of the leu and ilv genes (P1 and P2) or independently under the control of its own promoter (P3). In this paper we show that the production of ALDC is limited by two mechanisms. First, the strength of P3 decreases greatly during starvation for BCAAs and under other conditions that generally provoke the stringent response. Second, although aldB is actively transcribed from P1 and P2 during BCAA starvation, ALDC is not significantly produced from these transcripts. The aldB ribosome binding site (RBS) appears to be entrapped in a stem-loop, which is itself part of a more complex RNA folding structure. The function of the structure was studied by mutagenesis, using translational fusions with luciferase genes to assess its activity. The presence of the single stem-loop entrapping the aldB RBS was responsible for a 100-fold decrease in the level of aldB translation. The presence of a supplementary secondary structure upstream of the stem-loop led to an additional fivefold decrease of aldB translation. Finally, the translation of the ilvA gene terminating in the latter structure decreased the level of translation of aldB fivefold more, leading to the complete extinction of the reporter gene activity. Since three leucines and one valine are present among the last six amino acids of the ilvA product, we propose that pausing of the ribosomes during translation could modulate the folding of the messenger, as a function of BCAA availability. The purpose of the structure-dependent regulation could be to ensure the minimal production of ALDC required for the control of the acetolactate pool during BCAA synthesis but to avoid its overproduction, which would dissipate acetolactate. Large amounts of ALDC, necessary for operation of the acetoin pathway, could be produced under favorable conditions from the P3 transcripts, which do not contain the secondary structures.

Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) is a homofermentative bacterium that produces lactic acid during growth. We adapted the two-dimensional electrophoresis (2-DE) technique to study the response of this bacterium to acidity. De novo protein synthesis was monitored by [35S]methionine labeling of exponentially growing cultures under standard (pH 6) and acidic (pH 4.75) conditions. After 2-DE separation, the protein patterns were compared. The protein spots showing increased radioactivity levels under acid conditions were considered acid-induced. We determined the N-terminal amino acid sequence of three highly induced proteins; comparing these proteins to databases we identified them to be the well-known heat shock proteins GroES, GroEL, and DnaK. Their induction levels were measured and compared. This is the first study by 2-DE of stress response in L. bulgaricus. We established the method and present a protein map which will be useful for future studies.

In this work, we present evidence that indicates that RuvABC proteins resolve Holliday junctions in a way that prevents dimer formation in vivo. First, although arrested replication forks are rescued by recombinational repair in cells deficient for the Rep helicase, rep mutants do not require the XerCD proteins or the dif site for viability. This shows that the recombination events at arrested replication forks are generally not accompanied by the formation of chromosome dimers. Secondly, resolution of dimers into monomers is essential in the rep ruv strain because of an increased frequency of RecFOR recombination events in the chromosome of this mutant. This suggests that, in the absence of the Ruv proteins, chromosomal recombination leads to frequent dimerization. Thirdly, dif or xerC mutations increase the UV sensitivity of ruv-deficient cells 100-fold, whereas they do not confer UV sensitivity to ruv+ cells. This shows that recombinational repair of UV lesions is not accompanied by dimer formation provided that the RuvABC proteins are active. The requirement for dimer resolution in ruv strains is suppressed by the expression of the RusA Holliday junction resolvase; therefore, RusA also prevents dimer formation. We conclude that the inviability arising from a high frequency of dimer formation in rep or UV-irradiated cells is only observed in the absence of known enzymes that resolve Holliday junctions.

We studied DNA binding of a transcriptional repressor, CopF, displayed on a filamentous phage. Mutagenesis of a putative helix-turn-helix motif of CopF and of certain bases of the operator abolished the protein-DNA interaction, establishing the elements involved in CopF function and showing that phage display can be used to study repressor proteins.

The AddAB enzyme is important to homologous DNA recombination in Bacillus subtilis, where it is thought to be the functional counterpart of the RecBCD enzyme of Escherichia coli. In vivo, AddAB responds to a specific five-nucleotide sequence (5'-AGCGG-3' or its complement) in a manner analogous to the response of the RecBCD enzyme to interaction with chi sequences. Here, we show that purified AddAB enzyme is able to load at a double-stranded DNA end and is both a DNA helicase and nuclease, whose combined action results in the degradation of both strands of the DNA duplex. During translocation, recognition of the properly oriented sequence 5'-AGCGG-3' causes attenuation of the AddAB enzyme nuclease activity that is responsible for degradation of the strand 3'-terminal at the entry site. Therefore, we conclude that 5'-AGCGG-3' is the B. subtilis Chi site and it is hereafter referred to as chi(Bs). After encountering chi(Bs), both the degradation of the 5'-terminal strand and the helicase activity persist. Thus, processing of a double-stranded DNA end by the AddAB enzyme produces a duplex DNA molecule with a protruding 3'-terminated single-stranded tail, a universal intermediate of the recombination process., (Copyright 2000 Academic Press.)

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid-resistant insertional mutants of the L. lactis strain MG1363. Twenty-one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress-resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

The extracellular Staphylococcus hyicus lipase was expressed under the control of different promoters in Lactococcus lactis and Bacillus subtilis. Its expression at high and moderate levels is toxic for the former and the latter hosts, respectively. In L. lactis, the lipase was expressed at a high level, up to 30% of the total cellular proteins, under the control of the inducible promoter PnisA. About 80% of the lipase remained associated with the cells. Close to half of this amount remained associated with the inner side of the cytoplasmic membrane as unprocessed pre-pro-lipase. The other half was trapped by the cell wall and partially degraded at the N-terminal end. This result suggests that extracellular proteases degrade the lipase. Surprisingly, the kinetics and the pattern of lipase degradation were different in the two L. lactis subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis. The extracellular proteolytic systems that degrade lipase are thus different in these closely related subspecies. The incorrect export of the lipase is not due to an inappropriate leader peptide but may be due to an inefficiency of several steps of lipase secretion. We propose that (i) the S. hyicus lipase may require a special accessory system to be correctly exported or (ii) the kinetics of lipase synthesis may be a critical factor for proper folding.

The Escherichia coli UvrD helicase (or helicase II) is known for its involvement in DNA repair. We report that UvrD is required for DNA replication of several different rolling-circle plasmids in E. coli, whereas its homologue, the Rep helicase, is not. Lack of UvrD helicase does not impair the first step of plasmid replication, nicking of the double-stranded origin by the plasmid initiator protein. However, replication proceeds no further without UvrD. Indeed, the nicked plasmid molecules accumulate to a high level in uvrD mutants. We conclude that UvrD is the replicative helicase of various rolling-circle plasmids. This is the first description of a direct implication of UvrD in DNA replication in vivo.

Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able to degrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed of three enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. The transcription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand the physiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. We show that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown under anaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal. Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is not responsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pH nor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is able to degrade arginine under aerobiosis.

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.

Three asparagine synthetase genes, asnB, asnH, and asnO (yisO), were predicted from the sequence of the Bacillus subtilis genome. We show here that the three genes are expressed differentially during cell growth. In a rich sporulation medium, expression of asnB was detected only during exponential growth, that of asnH was drastically elevated at the transition between exponential growth and stationary phase, and that of asnO was seen only later in sporulation. In a minimal medium, both asnB and asnH were expressed constitutively during exponential growth and in stationary phase, while the expression of asnO was not detected in either phase. However, when the minimal medium was supplemented with asparagine, only the expression of asnH was partially repressed. Transcription analyses revealed that asnB was possibly cotranscribed with a downstream gene, ytnA, while the asnH gene was transcribed as the fourth gene of an operon comprising yxbB, yxbA, yxnB, asnH, and yxaM. The asnO gene is a monocistronic operon, the expression of which was dependent on one of the sporulation sigma factors, sigma-E. Each of the three genes, carried on a low-copy-number plasmid, complemented the asparagine deficiency of an Escherichia coli strain lacking asparagine synthetases, indicating that all encode an asparagine synthetase. In B. subtilis, deletion of asnO or asnH, singly or in combination, had essentially no effect on growth rates in media with or without asparagine. In contrast, deletion of asnB led to a slow-growth phenotype, even in the presence of asparagine. A strain lacking all three genes still grew without asparagine, albeit very slowly, implying that B. subtilis might have yet another asparagine synthetase, not recognized by sequence analysis. The strains lacking asnO failed to sporulate, indicating an involvement of this gene in sporulation.

In recD sbcB sbcD mutants, repair of UV-irradiated DNA is strongly RecF dependent, indicating that RecBC is inactive. This finding suggests that exonuclease V, exonuclease I (SbcB), and the SbcCD nuclease play a redundant role in vivo, which is essential for the recombination activity of the RecBC complex during UV repair.

Replication slippage is a particular type of error caused by DNA polymerases believed to occur both in bacterial and eukaryotic cells. Previous studies have shown that deletion events can occur in Escherichia coli by replication slippage between short duplications and that the main E. coli polymerase, DNA polymerase III holoenzyme is prone to such slippage. In this work, we present evidence that the two other DNA polymerases of E. coli, DNA polymerase I and DNA polymerase II, as well as polymerases of two phages, T4 (T4 pol) and T7 (T7 pol), undergo slippage in vitro, whereas DNA polymerase from another phage, Phi29, does not. Furthermore, we have measured the strand displacement activity of the different polymerases tested for slippage in the absence and in the presence of the E. coli single-stranded DNA-binding protein (SSB), and we show that: (i) polymerases having a strong strand displacement activity cannot slip (DNA polymerase from Phi29); (ii) polymerases devoid of any strand displacement activity slip very efficiently (DNA polymerase II and T4 pol); and (iii) stimulation of the strand displacement activity by E. coli SSB (DNA polymerase I and T7 pol), by phagic SSB (T4 pol), or by a mutation that affects the 3' --> 5' exonuclease domain (DNA polymerase II exo(-) and T7 pol exo(-)) is correlated with the inhibition of slippage. We propose that these observations can be interpreted in terms of a model, for which we have shown that high strand displacement activity of a polymerase diminishes its propensity to slip.

In addition to their essential catalytic role in protein biosynthesis, aminoacyl-tRNA synthetases participate in numerous other functions, including regulation of gene expression and amino acid biosynthesis via transamidation pathways. Herein, we describe a class of aminoacyl-tRNA synthetase-like (HisZ) proteins based on the catalytic core of the contemporary class II histidyl-tRNA synthetase whose members lack aminoacylation activity but are instead essential components of the first enzyme in histidine biosynthesis ATP phosphoribosyltransferase (HisG). Prediction of the function of HisZ in Lactococcus lactis was assisted by comparative genomics, a technique that revealed a link between the presence or the absence of HisZ and a systematic variation in the length of the HisG polypeptide. HisZ is required for histidine prototrophy, and three other lines of evidence support the direct involvement of HisZ in the transferase function. (i) Genetic experiments demonstrate that complementation of an in-frame deletion of HisG from Escherichia coli (which does not possess HisZ) requires both HisG and HisZ from L. lactis. (ii) Coelution of HisG and HisZ during affinity chromatography provides evidence of direct physical interaction. (iii) Both HisG and HisZ are required for catalysis of the ATP phosphoribosyltransferase reaction. This observation of a common protein domain linking amino acid biosynthesis and protein synthesis implies an early connection between the biosynthesis of amino acids and proteins.

Lactococcus lactis is an AT-rich gram positive bacterium phylogenetically close to the genus Streptococcus. Various strains of L. lactis are used in dairy industry as starters for cheese making. L. lactis is also one of the well characterized laboratory microorganisms, widely used for studies on physiology of lactic acid bacteria. We describe here a low redundancy sequence of the genome of the strain L. lactis IL1403. The strategy which we followed to determine the sequence consists of two main steps. First, a limited number of plasmids and lambda-phages that carry random segments of the genome were sequenced. Second, sequences of the inserts were used for production of novel sequencing templates by applying Multiplex Long Accurate PCR protocols. Using of these PCR products allowed to determine the sequence of the entire 2.35 Mb genome with a very low redundancy, close to 2. The error rate of the sequence is estimated to be below 1%. The correctness of the sequence assembly was confirmed by PCR amplification of the entire L. lactis IL1403 genome, using a set of 266 oligonucleotides. Anotation of the sequence was undertaken by using automatic gene prediction computer tools. This allowed to identify 1495 protein-encoding genes, to locate them on the genome map and to classify their functions on the basis of homology to known proteins. The function of about 700 genes expected to encode proteins that lack homologs in data bases cannot be reliably predicted in this way. The approach which we used eliminates high redundancy sequencing and mapping efforts, needed to obtain detailed and comprehensive genetic and physical maps of a bacterium. Availability of detailed genetic and physical maps of the L. lactis IL1403 genome provides many entries to study metabolism and physiology of bacteria from this group. The presence of 42 copies of five different IS elements in the IL1403 genome confirms the importance of these elements for genetic exchange in Lactococci. These include two previously unknown elements, present at seven and fifteen copies and designated IS1077 and IS983, respectively. Five potential or rudimentary prophages were identified in the genome by detecting clusters of phage-related genes. The metabolic and regulatory potential of L. lactis was evaluated by inspecting gene sets classified into different functional categories. L. lactis has the genetic potential to synthesise 20 standard amino acids, purine and pyrimidine nucleotides and at least four cofactors. Some of these metabolites, which are usually present in chemically defined media, can probably be omitted. About twenty compounds can be used by L. lactis as a sole carbon source. Some 83 regulators were revealed, indicating a regulatory potential close to that of Haemophilus influenzae, a bacterium with a similar genome size. Unexpectedly, L. lactis has a complete set of late competence genes, which may have concerted transcriptional regulation and unleadered polycistronic mRNAs. These findings open new possibilities for developing genetic tools, useful for studies of gene regulation in AT-rich gram positive bacteria and for engineering of new strains for the diary industry.

The genotypic and phenotypic diversity among isolates of the Lactobacillus curvatus/Lactobacillus graminis/Lactobacillus sakei group was evaluated by comparing RAPD data and results of biochemical tests, such as hydrolysis of arginine, D-lactate production, melibiose and xylose fermentation, and the presence of haem-dependent catalase. Analyses were applied to five type strains and to a collection of 165 isolates previously assigned to L. sakei or L. curvatus. Phenotypic and RAPD data were compared with each other and with previous DNA-DNA hybridization data. The phenotypic and genotypic separation between L. sakei, L. curvatus and L. graminis was clear, and new insights into the detailed structure within L. sakei and L. curvatus were obtained. Individual strains could be typed by RAPD and, after the elimination of similar or identical isolates, two sub-groups in both L. curvatus and L. sakei were defined. The presence or absence of catalase activity further distinguished the two L. curvatus sub-groups. By cloning and sequencing specific RAPD products, pairs of PCR primers were developed that can be used to specifically detect L. curvatus, L. sakei and each of the L. sakei sub-groups.

In Lactococcus lactis, the his operon contains all the genes necessary for histidine biosynthesis. It is transcribed from a unique promoter, localized 300 bp upstream of the first gene. The region corresponding to the untranslated 5' end of the transcript, named the his leader region, displays the typical features of the T box transcriptional attenuation mechanism which is involved in the regulation of many amino acid biosynthetic operons and tRNA synthetase genes in gram-positive bacteria. Here we describe the regulation of transcription of the his operon by the level of histidine in the growth medium. In the absence of histidine, two transcripts are present. One covers the entire operon, while the other stops at a terminator situated about 250 bp downstream of the transcription start point. DNA sequences implicated in regulation of the his operon were identified by transcriptional fusion with luciferase genes and site-directed mutagenesis. In addition to the previously defined sequences necessary for effective T-box-mediated regulation, new essential regions were identified. Eighteen percent of the positions of the his leader region were found to differ in seven distantly related strains of L. lactis. Analysis of the variable positions supports the folding model of the central part of the his leader region. Lastly, in addition to the T-box-mediated regulation, the operon is regulated at the level of initiation of transcription, which is repressed in the presence of histidine. An operator site, necessary for full repression, overlaps the terminator involved in the T box attenuation mechanism. The functionality of the operator is altered on plasmids with low and high copy numbers, suggesting that supercoiling may play a role in the expression of the his operon. The extents of regulation at the levels of initiation and attenuation of transcription are 6- to 8-fold and 14-fold, respectively. Together, the two levels of control allow a 120-fold range of regulation of the L. lactis operon by histidine.

Studies of cellular responses to stress conditions such as heat, oxygen or starvation have revealed the existence of numerous specific or interactive response pathways. We previously observed in Lactococcus lactis that inactivation of the recA gene renders the lactococcal strain sensitive not only to DNA-damaging agents but also to oxygen and heat. To further examine the stress response pathways in L. lactis, we isolated thermoresistant insertional mutants (Trm) of the recA strain. Eighteen independent trm mutations were identified and characterized. We found that mutations map in only seven genes, implicated in purine metabolism (deoB, guaA and tktA), phosphate uptake (pstB and pstS), mRNA stability (pnpA) and in one uncharacterized gene (trmA). All the trm mutations, with the exception of trmA, confer multiple stress resistance to the cell. Some of the mutations confer improved heat stress resistance not only in the recA but also in the wild-type context. Our results reveal that cellular metabolic pathways are intimately related to stress response and that the flux of particular metabolites, notably guanine and phosphate, may be implicated in stress response in lactococci.

Lactococcus lactis strains are widely used in industrial dairy fermentations. Conventional phenotypic tests have been used for years to classify members of this species into two subspecies, lactis and cremoris, and play a key role in the choice of strains to be used in particular cheese fermentations. DNA hybridisation techniques have also been used for strain classification, giving rise to two genome homology groups. However, results showed discrepancies between the two methods of classification. We applied the randomly amplified polymorphic DNA fingerprinting (RAPD) technique to resolve previous contradictions in lactococcal classifications. Unlike usual RAPD methods, we use three primers to classify 113 strains and integrate the resulting information by a digitised programme used for this purpose. Our analysis revealed three major RAPD groups, designated G1, G2 and G3. G1 and G3 contain strains of the lactis subspecies, and G2 contains strains of the cremoris subspecies, as previously defined by phenotypic characteristics. Moreover, group G1 corresponds to one genome homology group, and groups G2 and G3 correspond to the second one. The taxonomic structure within L. lactis is therefore unusual: two distinct genetic groups of strains show indistinguishable phenotypes, while conversely, two phenotypically distinct groups are genetically homologous. We hypothesize that a subfamily of the subsp. lactis group gave rise to the cremoris subspecies.

Replication arrest leads to the occurrence of DNA double-stranded breaks (DSB). We studied the mechanism of DSB formation by direct measure of the amount of in vivo linear DNA in Escherichia coli cells that lack the RecBCD recombination complex and by genetic means. The RuvABC proteins, which catalyze migration and cleavage of Holliday junctions, are responsible for the occurrence of DSBs at arrested replication forks. In cells proficient for RecBC, RuvAB is uncoupled from RuvC and DSBs may be prevented. This may be explained if a Holliday junction forms upon replication fork arrest, by annealing of the two nascent strands. RecBCD may act on the double-stranded tail prior to the cleavage of the RuvAB-bound junction by RuvC to rescue the blocked replication fork without breakage.

pAMbeta1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. It has been shown previously that initiation of pAMbeta1 replication requires a plasmid-encoded protein (RepE) and a short origin and is carried out by the host DNA polymerase I. It is not known which primer is used by this polymerase for initiating replication. Here, we report that a transcription fork passing through the origin is a limiting factor for plasmid replication. Transcription that activates the origin is initiated at the repE promoter and is thus regulated by the plasmid copy-number control system. Two lines of evidence suggest that the transcription generates the primer for the DNA polymerase I. First, the transcription must start upstream from the origin and progress in the direction of replication to be effective. Second, 3' ends of RNA transcripts initiated upstream of the origin map within the origin, provided that the Rep protein and an intact origin are present. This is the first report for simultaneous requirement of a transcription fork, a replication protein and the DNA polymerase I in initiation of DNA replication.

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.

Mutations affecting mismatch repair result in elevated frequencies of microsatellite length alteration in prokaryotes and eukaryotes. However, the finding that microsatellite instability is found often in cells with a functional mismatch repair system prompted a search for other factors of tract alteration. In the present report, we show that, in Escherichia coli, poly(AC/TG) tracts are destabilized by mutations that induce SOS. These observations may have implications for eukaryotic cells because recent results suggest the existence of a mammalian SOS response analogous to that in prokaryotes. In addition, a defect in the 5'-3' exonuclease domain of DNA polymerase I, homologous to the mammalian FEN1 and the yeast RAD27 nucleases, leads to a marked increase in repeat expansions characteristic of several genetic disorders. Finally, we found that the combination of a proofreading defect with mismatch repair deficiency results in extreme microsatellite instability.

The mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood. In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling-circle and a theta replicon. Genetic analyses revealed that the rolling-circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene. Inhibition is, however, independent of DNA synthesis at the theta origin. We conclude that rolling-circle inhibition is caused by an inhibitory signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.