A New Map Location for the ilvB Locus of ESCHERICHIA COLI

We isolated, in E. coli K12, new alleles of the ilvB locus, the structural gene for acetolactate synthase isoenzyme I, and showed them to map at or near the ilvB619 site. The map position of the ilvB locus was redetermined because plasmids containing the ilvC-cya portion of the chromosome did not complement mutations at the ilvB locus. Furthermore, diploids for the ilvEDAC genes formed with these plasmids in an ilvHI background facilitated the isolation of the new ilvB alleles. The ilvB locus was remapped and found to be located at 81.5 minutes, between the uhp and dnaA loci. This location was determined by two- and three-point transductional crosses, deletion mapping and complementation with newly isolated plasmids. One of the new alleles of the ilvB gene is a mu-1 insertion. When present in the donor strain, this allele interferes with the linkage of genes flanking the mu-1 insertion, as well as the linkage of genes to either side of the mu-1 insertion.     

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl₂ excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.     

Use of an EZ-Tn5-Based Random Mutagenesis System to Identify a Novel Toxin Regulatory Locus in Clostridium perfringens Strain 13

Background

Although useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.

Methodology/Principal Findings

An EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.

Conclusions/Significance

The new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.     

Identification of a Gene Regulating the Tissue Expression of a Phosphoglucomutase Locus in Rainbow Trout

Nine percent of the rainbow trout (Salmo gairdneri) from a hatchery source have a greater than 100-fold increase in expression of a phosphoglucomutase (PGM) locus, Pgm1, in the liver but have normal expression of this locus in other tissues. The results of genetic crosses are consistent with a single regulatory gene with additive inheritance being responsible for the differences in the amount of PGM activity in the liver.—The allele responsible for the expression of Pgm1 in the liver is apparently a recent mutation. This is supported by its restricted distribution in rainbow trout and the absence of liver Pgm1 expression in closely related species. This genetic system is valuable for future analysis of the control of gene expression and in determining the relative evolutionary importance of genetic variation at structural and regulatory genes.     

Genetic Analysis of Mating Locus Linked Mutations in CHLAMYDOMONAS REINHARDII

The mating-type (mt) locus of Chlamydomonas reinhardii has been analyzed using four mutant strains (imp-1, imp-10, imp-11 and imp-12). All have been shown, or are shown here, to carry mutations linked to either the plus (mt⁺) or the minus (mt^-) locus, and their behavior in complementation tests has allowed us to define several distinct functions for each locus. Specifically, we propose that the mt⁺ locus contains the following genes or regulatory elements: a locus designated sfu, which is necessary for sexual fusion between gametes; a locus designated upp (uniparental plus), which controls aspects of chloroplast gene inheritance and perhaps also zygote maturation; and a locus designated sad, which functions in sexual adhesion. The mt^- locus also contains a sad locus as well as a gene or regulatory element designated mid, which is necessary for the minus dominance in mt⁺/mt^- diploids.     

The Sinorhizobium meliloti Nutrient-Deprivation-Induced Tyrosine Degradation Gene hmgA Is Controlled by a Novel Member of the arsR Family of Regulatory Genes

The regulation of the nutrient-deprivation-induced Sinorhizobium meliloti homogentisate dioxygenase (hmgA) gene, involved in tyrosine degradation, was examined. hmgA expression was found to be independent of the canonical nitrogen regulation (ntr) system. To identify regulators of hmgA, secondary mutagenesis of an S. meliloti strain harboring a hmgA-luxAB reporter gene fusion (N4) was carried out using transposon Tn1721. Two independent Tn1721 insertions were found to be located in a positive regulatory gene (nitR), encoding a protein sharing amino acid sequence similarity with proteins of the ArsR family of regulators. NitR was found to be a regulator of S. meliloti hmgA expression under nitrogen deprivation conditions, suggesting the presence of a ntr-independent nitrogen deprivation regulatory system. nitR insertion mutations were shown not to affect bacterial growth, nodulation of Medicago sativa (alfalfa) plants, or symbiotic nitrogen fixation under the physiological conditions examined. Further analysis of the nitR locus revealed the presence of open reading frames encoding proteins sharing amino acid sequence similarities with an ATP-binding phosphonate transport protein (PhnN), as well as transmembrane efflux proteins.     

Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIA^Glc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIA^Glc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIA^Glc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIA^Glc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIA^Glc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.     

Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB^Man, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB^Man, IIC^Man, IID^Man, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB^Man as well as membrane fragments containing IIC^Man and IID^Man. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB^Man.     

A Discrete Transition Zone Organizes the Topological and Regulatory Autonomy of the Adjacent Tfap2c and Bmp7 Genes

Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.     

Genetic and metabolic regulation of purine base transport in Neurospora crassa.

Summary Neurospora crassa can utilize various purine bases such as xanthine or uric acid and their catabolic products as a nitrogen source. The early purine catabolic enzymes in this organism are regulated by induction and by ammonium repression. Studies were undertaken to investigate purine base transport and its regulation in Neurospora. The results of competition experiments with uric acid and xanthine transport strongly suggest that uric acid and xanthine share a common transport system. It was also shown that the common transport system for uric acid and xanthine is distinct from a second transport system shared by hypoxanthine, adenine and guanine, and apparently also distinct from the transport system(s) for adenosine, cytosine and uracil. Regulation of the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system was studied. The results reveal that the uric acid-xanthine transport system is regulated by ammonium repression, but does not require uric acid induction. Neither ammonium repression nor uric acid induction controls the hypoxanthine-adenine-guanine transport system. A gene, designated amr, which is believed to be a positive regulatory gene for nitrogen metabolism of Neurospora crassa, was found to dramatically affect both the uric acid-xanthine transport system and the hypoxanthine-adenine-guanine transport system. A model for the action of the amr locus as a positive regulatory gene and for the interaction between the amr gene product and its recognition sites will be discussed.     

Genetic control of nucleoside transport in sheep erythrocytes

Nucleoside transport in sheep erythrocytes is under the genetic control of two allelomorphic genes (Nu ^I and Nu ⁱ ), where Nu ^I codes for the functional absence of a high-affinity nucleoside transport system and is dominant to the gene (Nu ⁱ ) coding for the presence of the transport system. Kinetic and inhibitor experiments show that the high-affinity transport system is not present in heterozygous erythrocytes, demonstrating that the Nu ^I gene is completely dominant over the Nu ⁱ gene. It is suggested that the Nu locus may not represent the structural gene locus of the nucleoside transport system. Instead, it may be a regulator gene locus.     

Relationship between Monokaryotic Growth Rate and Mating Type in the Edible Basidiomycete Pleurotus ostreatus

The edible fungus Pleurotus ostreatus (oyster mushroom) is an industrially produced heterothallic homobasidiomycete whose mating is controlled by a bifactorial tetrapolar genetic system. Two mating loci (matA and matB) control different steps of hyphal fusion, nuclear migration, and nuclear sorting during the onset and progress of the dikaryotic growth. Previous studies have shown that the segregation of the alleles present at the matB locus differs from that expected for a single locus because (i) new nonparental B alleles appeared in the progeny and (ii) there was a distortion in the segregation of the genomic regions close to this mating locus. In this study, we pursued these observations by using a genetic approach based on the identification of molecular markers linked to the matB locus that allowed us to dissect it into two genetically linked subunits (matBα and matBβ) and to correlate the presence of specific matBα and matA alleles with differences in monokaryotic growth rate. The availability of these molecular markers and the mating type dependence of growth rate in monokaryons can be helpful for marker-assisted selection of fast-growing monokaryons to be used in the construction of dikaryons able to colonize the substrate faster than the competitors responsible for reductions in the industrial yield of this fungus.     

IS6110 Transposition and Evolutionary Scenario of the Direct Repeat Locus in a Group of Closely Related Mycobacterium tuberculosis Strains

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome of M. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.     

Genetic Control of the 2-Keto-3-Deoxy-d-Gluconate Metabolism in Escherichia coli K-12: kdg Regulon

2-Keto-3-deoxy-gluconate (KDG), an intermediate of the hexuronate pathway in Escherichia coli K-12, is utilized as the sole carbon source only in strains derepressed for the specific KDG-uptake system. KDG is metabolized to pyruvate and glyceraldehyde-3-phosphate via the inducible enzymes KDG-kinase and 2-keto-3-deoxy-6-phosphate-gluconate (KDPG) aldolase. However, another inducible pathway, where the KDG is the branch point, has been demonstrated. Genetic studies of the KDG degradative pathway reported in this paper led to the location of KDG kinase-negative and pleiotropic constitutive mutations. The kdgK locus, presumably the structural gene of the kinase, occurs at min 69 and is co-transducible with xyl. The mutants, simultaneously constitutive for the uptake, kinase, and aldolase, define a kdgR locus at min 36 between the co-transducible markers kdgA and oldD. As to the nature of the control exerted by the kdgR product, we have shown the following. (i) Thermosensitive mutants of the kdgR locus are inducible at low temperature but derepressed at 42 C for the three operons—kdgT (transport system), kdgK, and kdgA (KDPG aldolase). (ii) The kdgR⁺ allele is dominant to the kdgR constitutive allele. (iii) A deletion in kdgA extending into the regulatory gene, kdgR, leads to a constitutive expression of the nondeleted operons—kdgT and kdgK. These properties demonstrate that the kdg regulon is negatively controlled by the kdgR product. It is presumed that differences in operator and in promotor structures could explain the strong decoordination, respectively, in the induction and catabolic repression, of these three enzymes activities.     

Overproduction of lysine by mutant strains of Escherichia coli with defective lysine transport systems

Mutants selected on the basis of their resistance to S-(β-aminoethyl)cysteine and overproduction of lysine were found to be defective in the lysine transport system. The overproduction of lysine was not due to mutation affecting either of the two regulatory enzymes aspartokinase and dihydrodipicolinic acid synthetase. Uptake of labeled lysine by the lysine-specific transport system was reduced to a negligible level, while uptake by the lysine, ornithine, arginine system was also affected. A hypothesis regarding the nature of these mutations and their effects on the regulation of lysine biosynthesis is discussed.     

The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi

The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi. This locus consists of two genes, pecS and pecM, divergently transcribed. Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS. Purification and characterization of PecS, expressed either from E. coli, from the wild-type E. chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties. Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity. Moreover, determination of the Kd_app for operator regions of the PecS protein, produced either by the wild-type E. chrysanthemi or by E. coli, reveals similar affinities. Thus, in E. coli, there is likely to be at least one other PecM-like protein able to cross-react with the E. chrysanthemi PecS protein.     

Domain Structure of the ATP-Binding-Cassette Protein MalK of Salmonella typhimurium as Assessed by Coexpressed Half Molecules and LacK′-′MalK Chimeras

ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of Salmonella typhimurium and Escherichia coli is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon. The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein. In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of S. typhimurium. Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a malK strain and displayed substantial regulatory activity. The same regulatory activity was obtained when malKC1 was expressed separately. These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities. To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK. Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity. This activity increased with the lengths of the MalK portions present in the chimeras. Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264. In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.