Horizontal transfer of a phosphatase gene as evidence for mosaic structure of the Salmonella genome.

The genomes of Escherichia coli and Salmonella typhimurium are similar with respect to base composition, chromosome size, and the order, orientation and spacing of genes, but differ with respect to some 29 'loops', regions unique to one species. To evaluate the genetic basis for the structure and organization of the enteric bacterial genomes, we examined the gene encoding a non-specific acid phosphatase (phoN) which maps to a loop at 96 min on the S.typhimurium chromosome. We detected atypical base composition, codon usage pattern and trinucleotide frequencies. The 1.4 kb region containing phoN had an overall base composition of 43% G+C, while the G+C content at the third positions of codons in the phoN reading frame is only 39%, much lower than the Salmonella chromosome which averages 52%. Non-specific acid phosphatase activity, assayed in 14 Gram-negative species, was detected only in Morganella morganii and Providencia stuartii, organisms with low genomic G+C contents. Upstream of the phoN gene in Salmonella is a sequence with high similarity to the oriT region of incFII plasmids, suggesting that the phoN gene, and perhaps the entire loop structure, was acquired by lateral transmission in a plasmid-mediated event.     

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.     

The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease

Biochemical, immunological, and sequence analyses demonstrated that the glucose permease of Bacillus subtilis, the glucose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, is a single polypeptide chain with a C-terminal Enzyme III-like domain. A flexible hydrophilic linker, similar in length and amino acid composition to linkers previously identified in other regulatory or sensory transducing proteins, functions to tether the Enzyme IIIGlc-like domain of the protein to the membrane-embedded Enzyme IIGlc. Evidence is presented demonstrating that the Enzyme IIIGlc-like domain of the glucose permease plays a dual role and functions in the transport and phosphorylation of both glucose and sucrose. The sucrose permease appears to lack a sucrose-specific Enzyme III-like domain or a separate, soluble IIIScr protein. Enzyme IIScr was capable of utilizing the IIIGlc-like domain of the glucose permease regardless of whether the IIIGlc polypeptide was provided as a purified, soluble protein, as a membrane-bound protein within the same membrane as Enzyme IIScr, or as a membrane-bound protein within membrane fragments different from those bearing Enzyme IIScr. These observations suggest that the IIIGlc-like domain is an autonomous structural unit that assumes a conformation independent of the hydrophobic, N-terminal intramembranal domain of Enzyme IIGlc. Preferential uptake and phosphorylation of glucose over sucrose has been demonstrated by both in vivo transport studies and in vitro phosphorylation assays. Addition of the purified IIIGlc-like domain strongly stimulated the phosphorylation of sucrose, but not that of glucose, in phosphorylation assays that contained the two sugars simultaneously. The results suggest that the preferential uptake of glucose over sucrose is determined by competition of the corresponding sugar-specific permeases for the common P approximately IIIGlc/Scr domain.     

Identification of the phosphocarrier protein enzyme IIIgut: essential component of the glucitol phosphotransferase system in Salmonella typhimurium.

The phosphoenolpyruvate-dependent phosphorylation of glucitol has been shown to require four distinct proteins in Salmonella typhimurium: two general energy-coupling proteins, enzyme I and HPr, and two glucitol-specific proteins, enzyme IIgut and enzyme IIIgut. The enzyme IIgut was solubilized from the membrane and purified about 100-fold, free of the other protein constituents of the phosphotransferase system. Enzyme IIIgut was found in both the soluble and the membrane fractions. The soluble enzyme IIIgut was purified to near homogeneity by gel filtration, hydroxylapatite chromatography, and hydrophobic chromatography on butylagarose. It was sensitive to parital inactivation by trypsin and N-ethylmaleimide, but was stable at 80 degrees C. The protein had an approximate molecular weight of 15,000. It was phosphorylated in the presence of phosphoenolpyruvate, enzyme I, and HPr, and this phosphoprotein was dephosphorylated in the presence of enzyme IIgut and glucitol. Antibodies were raised against enzyme IIIgut. Enzyme IIIglc and enzyme IIIgut exhibited no enzymatic or immunological cross-reactivity. Enzyme IIgut, enzyme IIIgut, and glucitol phosphate dehydrogenase activities were specifically induced by growth in the presence of glucitol. These results serve to characterize the glucitol-specific proteins of the phosphotransferase system in S. typhimurium.     

Allosteric regulation of glycerol kinase by enzyme IIIglc of the phosphotransferase system in Escherichia coli and Salmonella typhimurium.

The mechanism by which enzyme IIIglc of the bacterial phosphotransferase system regulates the activity of crystalline glycerol kinase from Escherichia coli has been studied, and the inhibitory effects have been compared with those produced by fructose-1,6-diphosphate. It was shown that the free, but not the phosphorylated, form of enzyme IIIglc inhibits the kinase. Mutants of Salmonella typhimurium were isolated which were resistant to inhibition by either enzyme IIIglc (glpKr mutants) or fructose-1,6-diphosphate (glpKi mutants), and each mutant type was shown to retain full sensitivity to inhibition by the other regulatory agent. Other mutants were fully or partially resistant to regulation by both agents. The two regulatory sites on the kinase are evidently distinct but must overlap or interact functionally. Kinetic analyses have revealed several mechanistic features of the regulatory interactions. (i) Inhibition by both allosteric regulatory agents is strongly pH dependent, with maximal inhibition occurring at ca. pH 6.5 under the assay conditions employed. (ii) Binding of enzyme IIIglc to glycerol kinase is also pH dependent, the Ki being near 4 microM at pH 6.0 but near 10 microM at pH 7.0. (iii) Whereas fructose-1,6-diphosphate inhibition apparently requires that the enzyme exist in a tetrameric state, both the dimer and the tetramer appear to be fully sensitive to enzyme IIIglc inhibition. (iv) Inhibition by enzyme IIIglc (like that by fructose-1,6-diphosphate) is noncompetitive with respect to both substrates. (v) The inhibitory responses of glycerol kinase to fructose-1, 6-diphosphate and enzyme IIIglc show features characteristic of positive cooperativity at low inhibitor concentration. (vi) Neither agent inhibits completely at high inhibitor concentration. (vii) Apparent negative cooperativity with respect to ATP binding is observed with purified E. coli glycerol kinase, with glycerol kinase in crude extracts of wild-type S. typhimurium cells, and with glpKr and glpKi mutant forms of glycerol kinase from S. typhimurium. These results serve to characterize the regulatory interactions which control the activity of glycerol kinase by fructose-1,6-diphosphate and by enzyme IIIglc of the phosphotransferase system.     

Tempo and mode of concerted evolution in the L1 repeat family of mice

A 300-bp DNA sequence has been determined for 30 (10 from each of three species of mice) random isolates of a subset of the long interspersed repeat family L1. From these data we conclude that members of the L1 family are evolving in concert at the DNA sequence level in Mus domesticus, Mus caroli, and Mus platythrix. The mechanism responsible for this phenomenon may be either duplicative transposition, gene conversion, or a combination of the two. The amount of intraspecies divergence averages 4.4%, although between species base substitutions accumulate at the rate of approximately 0.85%/Myr to a maximum divergence of 9.1% between M. platythrix and both M. domesticus and M. caroli. Parsimony analysis reveals that the M. platythrix L1 family has evolved into a distinct clade in the 10-12 Myr since M. platythrix last shared a common ancestor with M. domesticus and M. caroli. The parsimony tree also provides a means to derive the average half-life of L1 sequences in the genome. The rates of gain and loss of individual copies of L1 were estimated to be approximately equal, such that approximately one-half of them turn over every 3.3 Myr.      

Study of the mitotic and meiotic chromosomes of sotol (<i>Dasylirion cedrosanum</i> Trel.)

The genus Dasylirion is a group of plants typically present in the Chihuahuan Desert, perennial, with a dioecious sexual behavior and commonly called sotoles. This genus has been little studied from the biological point of view, and the bases of its reproductive response remain unknown. In this work we studied the chromosome number and meiotic response of Dasylirion cedrosanum in the county of Saltillo, Coahuila, located at the North East of Mexico. For the preparation of mitotic chromosomes, we used a technique based on enzymatic treatment with pectolyase and cellulase, as well as staining with acetocarmin dye. For the study of meiosis, male flower buds were collected, fixed and stained for analysis with the same dye. As a result, the gametic (n = x = 19) and somatic chromosome (2n = 38) numbers of D. cedrosanum are reported for the first time, being consistent with previous findings in other Dasylirion species, which points to a constant ploidy level across the genus. Variation was observed in the morphology and size of the somatic chromosomes, with types ranging from submetacentric to subtelocentric, and sizes oscillating in a range of 4.43 µm, with an average total length of 112.38 µm for the diploid chromosome complement. This shows that the chromosome complement of D. cedrosanom would belong to a 3B classification of Stebins, with a medium variation between chromosome lengths and low chromosome asymmetry. This variation indicates the feasibility of constructing a chromosome ideotype for this species. The meiotic chromosome pairing showed a chromosome behavior consistent with a disomic inheritance characteristic of a diploid species, with prevalence of ring and chain bivalents, typically without pairing abnormalities. Bivalent configurations in all cases were symmetrical.The normal and symmetrical meiotic pairing indicates a balanced production of gametes, and suggests the absence of heteromorphic sex determination.     

Biofilm-defective mutants of Bacillus subtilis

Many bacteria can adopt organized, sessile, communal lifestyles. The gram-positive bacterium, Bacillus subtilis,forms biofilms on solid surfaces and at air-liquid interfaces, and biofilm development is dependent on environmental conditions. We demonstrate that biofilm formation by B. subtilis strain JH642 can be either activated or repressed by glucose, depending on the growth medium used, and that these glucose effects are at least in part mediated by the catabolite control protein, CcpA. Starting with a chromosomal Tn917-LTV3 insertional library, we isolated mutants that are defective for biofilm formation. The biofilm defects of these mutants were observable in both rich and minimal media, and both on polyvinylchloride abiotic surfaces and in borosilicate tubes. Two mutants were defective in flagellar synthesis. Chemotaxis was shown to be less important for biofilm formation than was flagellar-driven motility. Although motility is known to be required for biofilm formation in other bacteria, this had not previously been demonstrated for B. subtilis. In addition, our study suggests roles for glutamate synthase, GltAB, and an aminopeptidase, AmpS. The loss of these enzymes did not decrease growth or cellular motility but had dramatic effects on biofilm formation under all conditions assayed. The effect of the gltAB defect on biofilm formation could not be due to a decrease in poly-gamma-glutamate synthesis since this polymer proved to be nonessential for robust biofilm formation. High exogenous concentrations of glutamate, aspartate, glutamine or proline did not override the glutamate synthase requirement. This is the first report showing that glutamate synthase and a cytoplasmic aminopeptidase play roles in bacterial biofilm formation. Possible mechanistic implications and potential roles of biofilm formation in other developmental processes are discussed.     

Lysophospholipid flipping across the Escherichia coli inner membrane catalyzed by a transporter (LplT) belonging to the major facilitator superfamily

The transfer of phospholipids across membrane bilayers is protein-mediated, and most of the established transporters catalyze the energy-dependent efflux of phospholipids from cells. This work identifies and characterizes a lysophospholipid transporter gene (lplT, formally ygeD) in Escherichia coli that is an integral component in the 2-acylglycerophosphoethanolamine (2-acyl-GPE) metabolic cycle for membrane protein acylation. The lplT gene is adjacent to and in the same operon as the aas gene, which encodes the bifunctional enzyme 2-acyl-GPE acyltransferase/acyl-acyl carrier protein synthetase. In some bacteria, acyltransferase/acyl-ACP synthetase (Aas) and LplT homologues are fused in a single polypeptide chain. 2-Acyl-GPE transport to the inside of the cell was assessed by measuring the Aas-dependent formation of phosphatidylethanolamine. The Aas-dependent incorporation of [3H]palmitate into phosphatidylethanolamine was significantly diminished in deltalplT mutants, and the LplT-Aas transport/acylation activity was independent of the proton motive force. The deltalplT mutants accumulated acyl-GPE in vivo and had a diminished capacity to transport exogenous 2-acylglycerophosphocholine into the cell. Spheroplasts prepared from wild-type E. coli transported and acylated fluorescent 2-acyl-GPE with an apparent K(d) of 7.5 microM, whereas this high-affinity process was absent in deltalplT mutants. Thus, LplT catalyzes the transbilayer movement of lysophospholipids and is the first example of a phospholipid flippase that belongs to the major facilitator superfamily.