ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Adenosine Triphosphatases/biosynthesis , Bacterial Proteins/biosynthesis , Copper/metabolism , Lactic Acid/pharmacology , Lactobacillus/drug effects , Metals, Heavy/metabolism , Adaptation, Physiological/drug effects , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Cation Transport Proteins , Copper/pharmacology , Copper Transport Proteins , Copper-Transporting ATPases , Culture Media , Enzyme Induction , Hydrogen-Ion Concentration , Lactobacillus/enzymology , Lactobacillus/growth & development , Lactobacillus/physiology , Metals, Heavy/pharmacology , Molecular Sequence Data , Transcription, GeneticABSTRACT
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.
Subject(s)
Base Sequence , Evolution, Molecular , Genome, Bacterial , Lactobacillus delbrueckii/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbohydrate Metabolism , Interspersed Repetitive Sequences , Lactobacillus delbrueckii/metabolism , Molecular Sequence Data , Sequence Analysis, DNA , Streptococcus thermophilus/metabolism , Synteny , Yogurt/microbiologyABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , DNA Replication , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/metabolism , Bacillus subtilis/chemistry , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Genetic Complementation Test , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , Point Mutation , Protein Interaction Mapping , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/geneticsABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , Genes, Bacterial , Bacillus subtilis/cytology , Bacillus subtilis/metabolism , Cell Division/genetics , Cell Membrane/genetics , Coenzymes/genetics , Coenzymes/metabolism , Energy Metabolism/genetics , Genome, Bacterial , Mutation , Nucleotides/genetics , Nucleotides/metabolism , PhylogenyABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , Genetic Vectors/genetics , Plasmids/genetics , Replicon , Amino Acid Sequence , Bacillus subtilis/isolation & purification , Bacterial Proteins/genetics , Cloning, Molecular , DNA-Binding Proteins/genetics , Gene Dosage , Genetic Engineering/methods , Molecular Sequence Data , Replicon/genetics , Republic of Belarus , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Soil MicrobiologyABSTRACT
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.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , DNA Helicases/metabolism , Molecular Sequence Data , Sequence Analysis, Protein , Two-Hybrid System TechniquesABSTRACT
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.
Subject(s)
Bacillus subtilis/enzymology , DNA Polymerase III/metabolism , DNA Replication , DNA-Directed DNA Polymerase/metabolism , Genes, Essential/genetics , Bacillus subtilis/genetics , Bacillus subtilis/growth & development , Bacterial Proteins/biosynthesis , Chromosomes, Bacterial/genetics , DNA Polymerase III/genetics , DNA Repair , DNA Replication/genetics , DNA, Bacterial/biosynthesis , DNA-Directed DNA Polymerase/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Genes, Bacterial/genetics , Genome, Bacterial , Holoenzymes/genetics , Holoenzymes/metabolism , Mutation/genetics , RNA, Bacterial/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
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.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , Base Sequence , Biopolymers , DNA Primers , DNA Replication , DNA, Bacterial , DNA, Single-Stranded/metabolismABSTRACT
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.
Subject(s)
Bacillus subtilis/metabolism , Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA Replication , Escherichia coli Proteins , Bacillus subtilis/genetics , Bacterial Proteins/genetics , DNA Helicases/genetics , DNA Primase/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DnaB Helicases , Mutation , Replication Protein A , Suppression, GeneticABSTRACT
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.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , Geobacillus stearothermophilus/enzymology , Mutagenesis/genetics , Pyrococcus/enzymology , Thermococcus/enzymology , Thermus/enzymology , Artifacts , Base Sequence , DNA Replication/drug effects , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Enzyme Stability , Humans , Magnesium/pharmacology , Models, Genetic , Mutagenesis/drug effects , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA, Ribosomal, 18S/chemistry , RNA, Ribosomal, 18S/genetics , Sequence Deletion/genetics , Templates, GeneticABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacterial Proteins/metabolism , Growth Inhibitors/metabolism , Lactobacillus/metabolism , Anti-Bacterial Agents/pharmacology , Antibiosis , Bacterial Proteins/pharmacology , Cell Culture Techniques , Growth Inhibitors/pharmacology , Hydrogen Peroxide/pharmacology , Lactobacillus/classification , Lactobacillus/growth & development , Microbial Sensitivity Tests , Streptococcus/drug effects , TemperatureABSTRACT
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.
Subject(s)
Amino Acids, Branched-Chain/metabolism , Bacterial Proteins/metabolism , DNA-Binding Proteins , Lactococcus lactis/metabolism , Repressor Proteins/metabolism , Transcription, Genetic , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Dipeptides/chemistry , Dipeptides/metabolism , Gene Expression Regulation, Bacterial , Lactococcus lactis/genetics , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , Molecular Sequence Data , Mutation , Operon , Repressor Proteins/genetics , Sequence Homology, Amino AcidABSTRACT
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.
Subject(s)
Genome, Bacterial , Lactococcus lactis/genetics , Amino Acids/biosynthesis , Amino Acids/genetics , Amino Acids/metabolism , Bacterial Proteins/metabolism , Bacteriophages/genetics , Biological Transport, Active/genetics , Cell Wall/genetics , Cell Wall/metabolism , DNA Transposable Elements/genetics , Endopeptidases/genetics , Energy Metabolism/genetics , Enterobacteriaceae/genetics , Gene Expression Regulation/genetics , Gene Transfer, Horizontal/genetics , Lactic Acid/metabolism , Lactococcus lactis/isolation & purification , Lactococcus lactis/metabolism , Lactococcus lactis/virology , Molecular Sequence Data , Multigene Family/genetics , Nucleotides/biosynthesis , Nucleotides/genetics , Open Reading Frames/genetics , Proviruses/genetics , RNA, Bacterial/genetics , Sequence Analysis, DNA/methods , Signal Transduction/genetics , Transformation, Genetic/genetics , Vitamins/biosynthesis , Vitamins/geneticsABSTRACT
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.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase/metabolism , Recombination, Genetic , Binding Sites , DNA Polymerase III/metabolism , Models, Genetic , Repetitive Sequences, Nucleic Acid , Viral Proteins/metabolismABSTRACT
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.
Subject(s)
Gene Expression Regulation, Bacterial , Lactococcus lactis/genetics , Peptide Hydrolases/genetics , Peptides/metabolism , Repressor Proteins/metabolism , Artificial Gene Fusion , Blotting, Northern , Caseins/metabolism , Caseins/pharmacology , Culture Media , Dipeptides/metabolism , Genes, Reporter , Hot Temperature , Lactococcus lactis/metabolism , Luciferases/genetics , Luciferases/metabolism , Operon , Peptide Hydrolases/metabolism , Promoter Regions, Genetic , Protein Hydrolysates/metabolism , Protein Hydrolysates/pharmacologyABSTRACT
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.
Subject(s)
DNA Helicases/physiology , DNA-Binding Proteins , Escherichia coli Proteins , HSP70 Heat-Shock Proteins/physiology , Rec A Recombinases/genetics , Trans-Activators/physiology , DNA Replication , Escherichia coli/growth & development , Hydrogen Peroxide/toxicity , Mutation , Oxidation-ReductionABSTRACT
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.
Subject(s)
Bacillus subtilis/genetics , DNA Replication/genetics , Rec A Recombinases/metabolism , Recombination, Genetic/genetics , Tandem Repeat Sequences/genetics , Bacillus subtilis/drug effects , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Deletion , Gene Expression Regulation, Bacterial , Kanamycin Resistance/genetics , Mutation , Rec A Recombinases/geneticsABSTRACT
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.
Subject(s)
Bacteriophages/genetics , Lactococcus lactis/virology , DNA, Bacterial/genetics , DNA, Viral/chemistry , DNA, Viral/genetics , Gene Order , Genes, Viral/genetics , Genome, Bacterial , Genome, Viral , Lactococcus lactis/genetics , Lysogeny , Molecular Sequence Data , Open Reading Frames , Sequence Analysis, DNAABSTRACT
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.