The lactose carrier of Escherichia coli functionally incorporated in Rhodopseudomonas sphaeroides obeys the regulatory conditions of the phototrophic bacterium

Rhodopseudomonas sphaeroides was provided with the ability to transport lactose via conjugation with a strain of Escherichia coli bearing a plasmid containing the lactose operon (including the lac Y gene, coding for the lactose carrier or M protein) and subsequent expression of the lac operon in Rps. sphaeroides (Nano, F.E. and Kaplan, S. submitted). The initial rate of lactose transport in Rps. sphaeroides was studied as a function of the light intensity and the magnitude of the proton-motive force. The results demonstrate that lactose transport is regulated by the rate of cyclic electron transfer in the same way as the endogenous transport systems.     

Phylogeny and biogeography of the American live oaks (Quercus subsection Virentes): a genomic and population genetics approach

The nature and timing of evolution of niche differentiation among closely related species remains an important question in ecology and evolution. The American live oak clade, Virentes, which spans the unglaciated temperate and tropical regions of North America and Mesoamerica, provides an instructive system in which to examine speciation and niche evolution. We generated a fossil‐calibrated phylogeny of Virentes using RADseq data to estimate divergence times and used nuclear microsatellites, chloroplast sequences and an intron region of nitrate reductase (NIA‐i3) to examine genetic diversity within species, rates of gene flow among species and ancestral population size of disjunct sister species. Transitions in functional and morphological traits associated with ecological and climatic niche axes were examined across the phylogeny. We found the Virentes to be monophyletic with three subclades, including a southwest clade, a southeastern US clade and a Central American/Cuban clade. Despite high leaf morphological variation within species and transpecific chloroplast haplotypes, RADseq and nuclear SSR data showed genetic coherence of species. We estimated a crown date for Virentes of 11 Ma and implicated the formation of the Sea of Cortés in a speciation event ~5 Ma. Tree height at maturity, associated with fire tolerance, differs among the sympatric species, while freezing tolerance appears to have diverged repeatedly across the tropical–temperate divide. Sympatric species thus show evidence of ecological niche differentiation but share climatic niches, while allopatric and parapatric species conserve ecological niches, but diverge in climatic niches. The mode of speciation and/or degree of co‐occurrence may thus influence which niche axis plants diverge along.     

In silico evolved lac operons exhibit bistability for artificial inducers, but not for lactose

Bistability in the lac operon of Escherichia coli has been widely studied, both experimentally and theoretically. Experimentally, bistability has been observed when E. coli is induced by an artificial, nonmetabolizable, inducer. However, if the lac operon is induced with lactose, the natural inducer, bistability has not been demonstrated. We derive an analytical expression that can predict the occurrence of bistability both for artificial inducers and lactose. We find very different conditions for bistability in the two cases. Indeed, for artificial inducers bistability is predicted, but for lactose the condition for bistability is much more difficult to satisfy. Moreover, we demonstrate that in silico evolution of the lac operon generates an operon that avoids bistability with respect to lactose, but does exhibit bistability with respect to artificial inducers. The activity of this evolved operon strikingly resembles the experimentally observed activity of the operon. Thus our computational experiments suggest that the wild-type lac operon, which regulates lactose metabolism, is not a bistable switch. Nevertheless, for engineering purposes, this operon can be used as a bistable switch with artificial inducers.     

Interaction of Shigella sonnei phase-I strains carrying F'- or R386 plasmids with continuous HeLa- and L-line cells

The region of S. sonnei chromosome, located to the left of the gene lac I, has been found to be linked with the capacity of these bacteria for penetrating epithelial cells: this capacity is sharply suppressed in transconjugates carrying plasmids F' which cover the above-mentioned chromosomal region in recipients. The loss of virulence by transconjugates with transferred plasmids F'lac is not linked with the transfer of F factor proper, as those transconjugates which have acquired plasmids F' from E. coli donor strains K = 12 X 363 or F'his 131, not covering the lactose region to the left of the gene lac I, retain their virulence. The transfer of plasmid R 386 having no analogs of bacterial chromosomal genes leads to the loss of virulence due to the oss of the invasive capacity of bacteria.     

A novel phylogenetic clade of picocyanobacteria from the Mazurian lakes (Poland) reflects the early ontogeny of glacial lakes

The community of picocyanobacteria inhabiting the Great Mazurian Lakes system (comprising lakes ranging from mesotrophic to hypertrophic) is dominated by phycoerythrin-rich cells, which outnumber phycocyanin-rich cells, even in hypertrophic lakes. The genetic diversity and phylogeny of 43 strains of picocyanobacteria isolated from four Mazurian lakes were studied by analyzing the nucleotide sequences of the 16S rRNA gene and cpcBA-IGS operon. Phylogenetic analyses assigned some of the strains to several previously described clusters (Groups A, B, C, E and I) and revealed the existence of a novel clade, Group M (Mazurian), which exhibited a low level of similarity to the other clusters. Both phycocyanin and phycoerythrin picocyanobacteria were assigned to this clade based on an analysis of the 16S rRNA gene. The cpcBA sequence analysis assigned only phycocyanin strains to Group M, whereas the phycoerythrin strains from the M ribogroup were assigned to Groups B and E. We hypothesize that Group M originally contained only phycocyanin picocyanobacteria. The phycoerythrin found in strains belonging to ribogroup M seems to have been acquired through horizontal gene transfer as an adaptation to the changing environment early in the ontogeny of these glacial lakes.     

Glucose-lactose diauxie in Escherichia coli

Growth of Escherichia coli in medium containing glucose, at a concentration insufficient to support full growth, and containing lactose, is diauxic. A mutation in the gene, CR, which determines catabolite repression specific to the lac operon, was found to relieve glucose-lactose but not glucose-maltose diauxie. Furthermore, a high concentration of lactose was shown to overcome diauxie in a CR(+) strain. Studies on the induction of beta-galactosidase by lactose suggested that glucose inhibits induction by 10(-2)m lactose. Preinduction of the lac operon was found to overcome this effect. The ability of glucose to prevent expression of the lac operon by reducing the internal concentration of inducer as well as by catabolite repression is discussed.     

A mutant Ebg enzyme that converts lactose into an inducer of the lac operon.

Lactose is not itself an inducer of the lac operon, nor is it converted to an inducer by ebg+ beta-galactosidase of Escherichia coli. We report here the isolation of a mutant Ebg beta-galactosidase which is capable of converting lactose into an inducer of the lac operon.     

Inversion in the lactose region of Escherichia coli K-12.

A spontaneous mutant of Escherichia coli K-12, strain SY99, with an inversion in the lactose region was isolated and partially characterized. The inversion was detected due to inverse chromosomal conjugational transfer after introduction of an F42 (F'lac) episome. The termini of the inversion are between proAB and lac on one side and lac and proC on the other. The inverse conjugational transfer in SY99 did not appear to be absolute but was always accompanied by a residual "normal" counterclockwise mobilization. This residual transfer was further shown to be caused by the intrinsic instability of this region (at least in the line W3110). The possible involvement of IS3 elements flanking the lactose operon is discussed.     

Rampant horizontal transfer and duplication of rubisco genes in eubacteria and plastids

Previous work has shown that molecular phylogenies of plastids, cyanobacteria, and proteobacteria based on the rubisco (ribulose-1,5- bisphosphate carboxylase/oxygenase) genes rbcL and rbcS are incongruent with molecular phylogenies based on other genes and are also incompatible with structural and biochemical information. Although it has been much speculated that this is the consequence of a single horizontal gene transfer (of a proteobacterial or mitochondrial rubisco operon into plastids of rhodophytic and chromophytic algae), neither this hypothesis nor the alternative hypothesis of ancient gene duplication have been examined in detail. We have conducted phylogenetic analyses of all available bacterial rbcL sequences, and representative plastid sequences, in order to explore these alternative hypothesis and fully examine the complexity of rubisco gene evolution. The rbcL phylogeny reveals a surprising number of gene relationships that are fundamentally incongruent with organismal relationships as inferred from multiple lines of other molecular evidence. On the order of six horizontal gene transfers are implied by the form I (L8S8) rbcL phylogeny, two between cyanobacteria and proteobacteria, one between proteobacteria and plastids, and three within proteobacteria. Alternatively, a single ancient duplication of the form I rubisco operon, followed by repeated and pervasive differential loss of one operon or the other, would account for much of this incongruity. In all probability, the rubisco operon has undergone multiple events of both horizontal gene transfer and gene duplication in different lineages.      

Enzymatic adaptation by bacteria under pressure.

A study of enzymic adaptation under hydrostatic pressure by moderately barotolerant bacteria that can grow at pressure up to about 500 atm revealed that some adaptive processes are relatively insensitive to pressure, whereas others are sufficiently barosensitive to compromise survival capacity in situations requiring adaptation to new substrates under pressure. Examples of the former include adaptation of Escherichia coli to arabinose catabolism for growth and adaptation of Streptococcus faecalis to catabolism of lactose, ribose, or maltose. Examples of the latter include derepression of the lac operon in Escherichia coli and induction of penicillinase synthesis by Bacillus licheniformis. For both these barosensitive systems, pressure had little effect on enzyme levels in constitutive strains or in bacteria that had previously been induced at 1 atm. Moreover, it had no detectable effect on penicillinase secretion. However, pressures of 300 to 400 atm were found to reduce markedly rates and extents of enzyme synthesis by bacteria undergoing derepression or adaptation. This inhibitory effect of pressure was reflected in greater barosensitivity with extended lag and slower growth of initially unadapted Escherichia coli cells inoculated into minimal medium with lactose as sole source of carbon and fuel, and by major reductions in the minimal inhibitory concentrations of penicillin G for unadapted B. licheniformis cells inoculated into complex, antibiotic-containing media. Cyclic adenosine 5'-monophosphate did not reverse pressure inhibition of derepression of the lac operon, and catabolite repression was complete under pressure. However, derepression of the lac operon was more sensitive to pressure at low concentrations of inducer than at high concentrations. Apparent volume changes for derepression were 94 and 60 ml/mol at inducer concentrations of about 0.5 and 5 mM, respectively. Pressure was found not to be inhibitory for uptake of beta-galactosides; in fact, it was somewhat stimulatory. Therefore, results were interpreted in terms of inducer binding and subsequent conversion of an operator-inducer-repressor complex to inactive repressor and operator. Both reactions appeared to result in an increase in volume, the former more so than the latter. We found also that 200 atm was actually stimulatory for growth of Escherichia coli in minimal media, and the bacterium was in a sense barophilic.     

NICHE AND RANGE SIZE PATTERNS SUGGEST THAT SPECIATION BEGINS IN SMALL,ECOLOGICALLY DIVERGED POPULATIONS IN NORTH AMERICAN MONKEYFLOWERS (MIMULUS SPP.)

Closely related species (e.g., sister taxa) often occupy very different ecological niches and can exhibit large differences in geographic distributions despite their shared evolutionary history. Budding speciation is one process that may partially explain how differences in niche and distribution characteristics may rapidly evolve. Budding speciation is the process through which new species form as initially small colonizing populations that acquire reproductive isolation. This mode of species formation predicts that, at the time of speciation, sister species should have highly asymmetrical distributions. We tested this hypothesis in North American monkeyflowers, a diverse clade with a robust phylogeny, using data on geographical ranges, climate, and plant community attributes. We found that recently diverged sister pairs have highly asymmetrical ranges and niche breadths, relative to older sister pairs. Additionally, we found that sister species occupy distinct environmental niche positions, and that 80% of sister species have completely or partially overlapping distributions (i.e., are broadly sympatric). Together, these results suggest that budding speciation has occurred frequently in Mimulus, that it has likely taken place both inside the range and on the range periphery, and that observed divergences in habitat and resource use could be associated with speciation in small populations.     

The Evolutionary History of the Transposable Element Penelope in the Drosophila virilis Group of Species

We have used phylogenetic techniques to study the evolutionary history of the Penelope transposable element in the Drosophila virilis species group. Two divergent types of Penelope have been detected, one previously described, clade I, and a new one which we have termed clade III. The phylogeny of some copies of the Penelope clade I element was partially consistent with the species phylogeny of the D. montana subphylad, suggesting cospeciation and allowing the estimation of the evolutionary rate of Penelope. Divergence times of elements found in different species are younger than the age of the species, suggesting horizontal transfer events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Dmitri Petrov]     

Activation of quiescent infections by postharvest pathogens during transition from the biotrophic to the necrotrophic stage

The evolution of bacterial pathogens from nonpathogenic ancestors is marked principally by the acquisition of virulence gene clusters on plasmids and pathogenicity islands via horizontal gene transfer. The flip side of this evolutionary force is the equally important adaptation of the newly minted pathogen to its new host niche. Pathoadaptive mutations take the form of modification of gene expression such that the pathogen is better fit to survive within the new niche. This mini-review describes the concept of pathoadaptation by loss of gene function. In this process, genes that are no longer compatible with the novel lifestyle of the pathogen are selectively inactivated either by point mutation, insertion, or deletion. These genes are called 'antivirulence genes'. Selective pressure sometimes leads to the deletion of large regions of the genome that contain antivirulence genes generating 'black holes' in the pathogen genome. Inactivation of antivirulence genes leads to a pathogen that is highly adapted to its host niche. Identification of antivirulence genes for a particular pathogen can lead to a better understanding of how it became a pathogen and the types of genetic traits that need to be silenced in order for the pathogen to colonize its new host niche successfully.     

Transport regulation of recombinant gene expression in E. coli and B. subtilis

Expression kinetics of the lactose (lac) operon in Escherichia coli are reviewed for both wild-type and recombinant cell cultures under chemostatic conditions. A unified model which involves regulation of active inducer (lactose) transport, promoter-operator regulated expression of the lac operon, glucose-mediated inducer exclusion, and catabolite repression is summarized and supporting data is shown to verify its accuracy. The synthesis of alpha-amylase with a recombinant form of Bacillus subtilis is also reviewed to point out generic features in transport regulation, the lac operon model providing a point of departure. While there are many similarities in the influence of transport on both regulating models, there are also important differences. In a chemostat system, the synthesis of alpha-amylase is nongrowth associated, while beta-galactosidase is a growth-associated enzyme. Nevertheless, transport regulation is an important feature in both instances.     

Selective inhibition of carbohydrate transport by the local anesthetic procaine in Escherichia coli.

Maltose and lactose transport systems have been used to investigate the action of procaine on insertion and activity of membrane proteins and translocation of exported proteins in Escherichia coli. Procaine mildly inhibited growth on lactose. The level of inhibition was consistent with the small reduction observed in active and facilitated transport functions of the lac permease. However, procaine caused a severe reduction of growth rate on maltose, as well as an inhibition of induction of maltose regulon activities. In both constitutive and inducible strains, the synthesis of both maltose transport activity (malB operon) and amylomaltase activity (malA operon) was inhibited. Coordinate inhibition of soluble and membrane products was not observed with the lac operon. beta-Galactosidase synthesis proceeded normally during growth on procaine, whereas, the appearance of new transport activity was reduced. Regardless of carbon source, procaine specifically inhibited the appearance of ompF protein in the membrane fraction.     

Use of bio-lac fusion strains to study regulation of biotin biosynthesis in Escherichia coli.

The technique developed by Casadaban (M. J. Casadaban, J. Mol. Biol. 104: 541-555, 1976) has been employed to construct Escherichia coli K-12 derivatives in which the genes determining lactose utilization are fused to the regulatory region of the biotin operon. Fusions of the lac genes to either arm of this divergently transcribed operon have been isolated. When the operon is derepressed, expression of the lac genes is sufficient to permit growth on lactose minimal medium. Repressing conditions prevent growth on lactose. This property of bio-lac fusion strains, as well as the ease of determining the level of operon expression by assaying beta-galactosidase, was used for the isolation and characterization of mutants defective in repression. Preliminary analyses of several newly isolated regulatory mutants are presented. For the several birA mutants examined, there appeared to be no direct correlation between effects on minimum biotin requirement and alterations in repressibility, suggesting a possible dual function for the gene. Parallel attempts to obtain fusions of lac to bioH were unsuccessful, indicating lack of direct biotin control at the bioH locus.     

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

Studies on beta-galactoside transport in a Proteus mirabilis merodiploid carrying an Escherichia coli lactose operon

Merodiploid derivatives bearing an F-linked lac operon (i(+), o(+), z(+), y(+), a(+)) from Escherichia coli were prepared from a Proteus mirabilis strain unable to utilize lactose and from a lac deletion strain of E. coli. A suitable growth medium was found in which the episomal element in the P. mirabilis derivative was sufficiently stable to allow induction of the episome-borne lac operon and thus to permit a comparison of the activities and properties of E. coli lac products in the intracellular environments of P. mirabilis and E. coli. In both derivatives the episomal lac operon was shown to be repressed in the absence of inducer. Kinetics of induction with gratuitous inducer (isopropyl-1-thio-beta-d-galactoside) were similar for both beta-galactosidase activity (beta-d-galactoside galactohydrolase, EC 3.4.1.23) and beta-galactoside transport activity in both derivatives, although the ratio of galactoside transport to beta-galactosidase activity was approximately 1.6-fold higher in the E. coli derivative. Comparison of beta-galactosidase and M-protein (lac y gene product)-specific activities indicated coordinate expression of the induced lac operon in both derivatives. Quantitatively, the maximal beta-galactosidase specific activity was two or three times higher for the E. coli derivative. A significant sodium azide inhibition (65% inhibition by 10 mM sodium azide) of lactose permease-mediated transport of o-nitrophenyl-beta-galactoside from an outside region of high concentration to an inside region of very low concentration ("downhill transport") was observed for the P. mirabilis derivative. Identical conditions for the E. coli derivative yielded only about 15% inhibition. Active transport of thiomethyl-beta-galactoside was similar for both derivatives, the major difference being that active transport was more sensitive to azide poisoning in the P. mirabilis derivative. Preliminary examination of the thiomethyl-beta-galactoside derivatives following active transport did not demonstrate the accumulation of a phosphorylated product in either strain but did reveal an unidentified derivative present in the P. mirabilis merodiploid extract which was not detectable in the E. coli merodiploid.