Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.     

Phosphorylation of Streptococcus salivarius lactose permease (LacS) by HPr(His ~ P) and HPr(Ser-P)(His ~ P) and effects on growth

The oral bacterium Streptococcus salivarius takes up lactose via a transporter called LacS that shares 95% identity with the LacS from Streptococcus thermophilus, a phylogenetically closely related organism. S. thermophilus releases galactose into the medium during growth on lactose. Expulsion of galactose is mediated via LacS and stimulated by phosphorylation of the transporter by HPr(His approximately P), a phosphocarrier of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). Unlike S. thermophilus, S. salivarius grew on lactose without expelling galactose and took up galactose and lactose concomitantly when it is grown in a medium containing both sugars. Analysis of the C-terminal end of S. salivarius LacS revealed a IIA-like domain (IIA(LacS)) almost identical to the IIA domain of S. thermophilus LacS. Experiments performed with purified proteins showed that S. salivarius IIA(LacS) was reversibly phosphorylated on a histidine residue at position 552 not only by HPr(His approximately P) but also by HPr(Ser-P)(His approximately P), a doubly phosphorylated form of HPr present in large amounts in rapidly growing S. salivarius cells. Two other major S. salivarius PTS proteins, IIAB(L)(Man) and IIAB(H)(Man), were unable to phosphorylate IIA(LacS). The effect of LacS phosphorylation on growth was studied with strain G71, an S. salivarius enzyme I-negative mutant that cannot synthesize HPr(His approximately P) or HPr(Ser-P)(His approximately P). These results indicated that (i) the wild-type and mutant strains had identical generation times on lactose, (ii) neither strain expelled galactose during growth on lactose, (iii) both strains metabolized lactose and galactose concomitantly when grown in a medium containing both sugars, and (iv) the growth of the mutant was slightly reduced on galactose.     

角质形成细胞生长因子(KGF)在喉粘膜良性、癌前及恶性病变中的mRNA水平分析

利用原位杂交的方法检测KGFmRNA在正常喉粘膜上皮（N）、慢性非特异性炎症（IF）、不典型增生（DYS）及鳞癌（SCC）中的转录水平,探讨KGF在喉粘膜良性及恶性病变中的分布和可能的作用。结果表明,KGFmRNA不仅在间质中的成纤维细胞中表达,少量的炎细胞及血管内皮细胞中亦表达,而且从N、IF、DYS到SCC、KGFmRNA转录水平逐渐增强;上皮细胞及肿瘤性上皮细胞不表达KGFmRNA,KGFmRNA在分化差的SCC周围间质中表达较分化好的SCC周围间质增多。结论：KGF在上皮与间充质细胞的交互作用中发挥着重要的作用,对维持喉粘膜正常结构、代谢及喉癌的发生发展具有重要意义。     

Effect of bacterial cell wall and lipopolysaccharide on arachidonic acid metabolism by caruncular and allantochorionic tissues from cows that calved normally and those that retained fetal membranes

Immunoactive eicosanoids may have a role in both placental separation and uterine involution in cattle. In the present study, we examined the effects of bacterial cell wall preparation and endotoxins on in vitro prostaglandin synthesis and arachidonic acid (AA) metabolism by caruncular and allantochorionic tissues. Placentomes were obtained about 6 h post partum from cows that delivered normally (n = 10) or those with retained fetal membranes (n = 4); the tissue explants were incubated for 6 h in the presence of labeled or nonlabeled AA. Prostaglandin F(2alpha) (PGF(2alpha)) and E(2) (PGE(2)) were measured by radioimmunoassay, and labeled AA metabolites were separated by reverse phase-high pressure-liquid chromatography. There was no effect of bacterial cell wall preparations or endotoxins on in vitro caruncular PGF(2alpha) secretion. However, bacterial products increased caruncular PGE(2) secretion in both cows that delivered normally and those with retained fetal membranes. For normal delivery cows caruncular tissue, bacterial product also increased leukotriene B(4) (LTB(4)) and decreased both thromboxane B(2) (TXB(2)) and hydroxy-eicosatetranoic acids (HETE) in vitro secretion. For the allantochorion, bacterial products increased in vitro PGF(2alpha) secretion only in cows that delivered normally and increased PGE(2) secretion essentially in cows with retained fetal membranes. In general, 6 keto PGF(1alpha) was the main metabolite secreted by both allantochorionic and carucular tissues. However, in cows with retained fetal membranes, PGE(2) became the most important metabolite secreted by allantochorion, especially in the presence of endotoxin. In conclusion, these results suggest that bacteria found in the early postpartum uterus or their endotoxin affect primarily caruncular and allantochorionic PGE(2) synthesis.     

Cotransformation and Targeted Gene Inactivation in the Maize Anthracnose Fungus, Glomerella graminicola

Cotransformation of Glomerella graminicola was achieved with the G. graminicola genes TUB1R1 (encoding a β-tubulin which confers resistance to the fungicide benomyl) and PYR1 (encoding orotate phosphoribosyl transferase, which confers pyrimidine prototrophy). The cotransformation frequency was about 30% when selection was for pyrimidine prototrophy (Pyr⁺) and 87% when selection was for benomyl-resistant (Bml^r) transformants. Southern blots confirmed that both transforming DNAs had integrated into the genomes of transformants which were expressing both Pyr⁺ and Bml^r phenotypes. A plasmid, p23, which contained a truncated 500-bp segment representing the central region of the PYR1 gene was constructed. The plasmid was introduced with pCG7, containing TUB1R1, into G. graminicola M1.001 (Pyr⁺ Bml^s), and Bml^r transformants were selected. The Bml^r transformants were screened on medium which did not contain uridine in order to identify Pyr^- mutants created by integration of p23 at the PYR1 locus. None of the primary transformants were Pyr^-, but 0.2% of uninucleate conidia collected from the pooled primary transformants gave rise to Pyr^- auxotrophs. Southern blots representing two of these Pyr^- mutants confirmed that they had the expected homologous integration of p23 at the PYR1 locus. This suggested that integration resulted in production of two nonfunctional copies of the gene, one lacking the 5′ sequences and the other lacking the 3′ sequences. This study demonstrates the feasibility of using cotransformation to perform targeted gene disruptions in G. graminicola.     

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.     

Expansion of the rare Eucalyptus risdonii under climate change through hybridization with a closely related species despite hybrid inferiority

Background and AimsHybridization is increasingly recognized as an integral part of the dynamics of species range expansion and contraction. Thus, it is important to understand the reproductive barriers between co-occurring species. Extending previous studies that argued that the rare Eucalyptus risdonii was expanding into the range of the surrounding E. amygdalina by both seed and pollen dispersal, we here investigate the long-term fitness of both species and their hybrids and whether expansion is continuing.MethodsWe assessed the survival of phenotypes representing a continuum between the two pure species in a natural hybrid swarm after 29 years, along with seedling recruitment. The performance of pure species as well as of artificial and natural hybrids was also assessed over 28 years in a common garden trial.Key ResultsIn the hybrid zone, E. amygdalina adults showed greater mortality than E. risdonii, and the current seedling cohort is still dominated by E. risdonii phenotypes. Morphologically intermediate individuals appeared to be the least fit. Similar results were observed after growing artificial first-generation and natural hybrids alongside pure species families in a common garden trial. Here, the survival, reproduction, health and growth of the intermediate hybrids were significantly less than those of either pure species, consistent with hybrid inferiority, although this did not manifest until later reproductive ages. Among the variable progeny of natural intermediate hybrids, the most E. risdonii-like phenotypes were the most fit.ConclusionsThis study contributes to the increasing number of reports of hybrid inferiority in Eucalyptus, suggesting that post-zygotic barriers contribute to the maintenance of species integrity even between closely related species. However, with fitness rapidly recovered following backcrossing, it is argued that hybridization can still be an important evolutionary process, in the present case appearing to contribute to the range expansion of the rare E. risdonii in response to climate change.     

Pollen dispersal from exotic eucalypt plantations

The introgression of genes from exotic species or populations into gene pools of native species is a widespread concern in agricultural systems. This is also an issue of increasing importance in forest systems as there has been a dramatic expansion of tree plantations, which have now reached 180 million ha globally. This has recently occurred in Australia with eucalypts. To help assess the risk of genetic pollution, we assess the pattern of realised pollen dispersal from exotic Eucalyptus nitens plantations into native E. ovata forest in Tasmania. We assessed the frequency of F₁ hybrids in open-pollinated seed collected from native E. ovata trees located at varying distance from three exotic E. nitens plantations in Tasmania. Over 119,000 seedlings were screened for morphological markers diagnostic of each species and the F₁ hybrid. F₁ hybridisation averaged 7.2% within 100 m of the exotic E. nitens, with one native tree reaching 56%, but diminished to 0.7% by 200–300 m and continued at this low level to the limits of the sampling at 1.6 km. The decay in the percentage of interspecific F₁ hybridisation with distance followed a power function with a negative exponent (%F₁ = 91.435distance^–0.789; R²=0.84). Eucalyptus nitens is exclusively pollinated by small insects (smaller than honeybees), which the study shows can disperse pollen over 1.6 km. However, the restriction of most exotic F₁ hybridisation to within 200 m of exotic plantations presents clear opportunities to manage the genetic impacts of plantations on native forests.     

Glacial refugia and reticulate evolution: the case of the Tasmanian eucalypts

Tasmania is a natural laboratory for investigating the evolutionary processes of the Quaternary. It is a large island lying 40-44 degrees S, which was repeatedly glaciated and linked to southeastern continental Australia during the Quaternary. Climate change promoted both the isolation of species in glacial refugia, and an exchange between Tasmanian and mainland floras. Eucalyptus is a complex and diverse genus, which has increased in abundance in Australia over the past 100 kyr, probably in response to higher fire frequency. Morphological evidence suggests that gene flow may have occurred between many eucalypt species after changes in their distribution during the Quaternary. This paper summarizes recent genetic evidence for migration and introgressive hybridization in Tasmanian Eucalyptus. Maternally inherited chloroplast DNA reveals a long-term persistence of eucalypts in southeastern Tasmanian refugia, coupled with introgressive hybridization involving many species. Detailed analysis of the widespread species Eucalyptus globulus suggests that migration from mainland Australia was followed by introgression involving a rare Tasmanian endemic. The data support the hypothesis that changes in distribution of interfertile species during the Quaternary have promoted reticulate evolution in Eucalyptus.