Mutations affecting the Shine-Dalgarno sequences of the untranslated region of the Escherichia coli gltBDF operon.

Individual mutations which affected each of the two Shine-Dalgarno sequences at the 5' untranslated region of the gltB gene of Escherichia coli were characterized. They were isolated in plasmids carrying a gltB'-'lacZ protein fusion preceded by the regulatory region of the gltBDF operon. Subcloning and nucleotide sequencing of approximately 1,206 bp of DNA encompassing the gltBDF regulatory region showed that the mutations affected the first base at each of the two identical Shine-Dalgarno sequences, SD1 and SD2, located 40 and 8 bases, respectively, upstream from the putative gltB open reading frame. Only mutation gltB2r227, an adenine in place of a guanine, affecting the first base of SD2, lowered beta-galactosidase expression significantly, i.e., about fivefold. The results suggest that SD2 is the preferred functional site at which ribosomes initiate gltB mRNA translation.     

Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis

The effects of water-soluble nonstarch polysaccharides (sNSP) on human metabolism are considered to be beneficial because they decrease postprandial glycaemia and insulinaemia following ingestion of starch-rich foods. The mechanisms by which sNSP attenuate the postprandial rise in blood glucose are not well understood but their presence increases the viscosity of gastrointestinal contents, which affects physiological functions, e.g. gastric emptying and peristalsis. Increased viscosity and decreased water activity during hydrothermal treatment of starch could influence alpha-amylase action.Using guar galactomannan as a representative of sNSP, we found that galactomannan has a direct noncompetitive inhibitory effect on alpha-amylase with a K(i) value of approximately 0.5% (3.3 microM). The inhibition is not time dependent and studies suggest direct binding of the enzyme to galactomannan; the resulting galactomannan-amylase complex being inactive. Processing of starch at low water levels greatly affects the catalytic efficiency of alpha-amylase. The Km value for starch heat treated in limited water is raised and kcat is lowered relative to starch gelatinised in excess water. Since galactomannan has no effect on the Km of alpha-amylase, we conclude that the inhibitory action of the polymer is not secondary to a decrease in available water. Neither does it seem to be a consequence of impaired diffusion of enzyme, substrate and products because of an increase in viscosity of the medium.Thus, the effects of sNSP in lowering postprandial glycaemia not only involve modifications of gut physiology, but also include direct inhibition of the first stage in the biochemical degradation of starch.     

Biochemical study of multiple CheY response regulators of the chemotactic pathway of Rhodobacter sphaeroides

The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac(32)P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.     

Full resistance of herpes simplex virus type 1-infected primary human cells to alpha interferon requires both the Us11 and gamma(1)34.5 gene products

The gamma(1)34.5 gene product is important for the resistance of herpes simplex virus type 1 (HSV-1) to interferon. However, since the inhibition of protein synthesis observed in cells infected with a gamma(1)34.5 mutant virus results from the combined loss of the gamma(1)34.5 gene product and the failure to translate the late Us11 mRNA, we sought to characterize the relative interferon sensitivity of mutants unable to produce either the Us11 or the gamma(1)34.5 polypeptide. We now demonstrate that primary human cells infected with a Us11 mutant virus are hypersensitive to alpha interferon, arresting translation upon entry into the late phase of the viral life cycle. Furthermore, immediate-early expression of Us11 by a gamma(1)34.5 deletion mutant is sufficient to render translation resistant to alpha interferon. Finally, we establish that the Us11 gene product is required for wild-type levels of replication in alpha interferon-treated cells and, along with the gamma(1)34.5 gene, is an HSV-1-encoded interferon resistance determinant.     

Biological control of waterhyacinth in Sinaloa Mexico with the weevils Neochetina eichhorniae and N. bruchi

Water hyacinth (Eichhornia crassipes (Mart.) Solms.)creates severe problems in the irrigationdistricts of Mexico, particularly in westernSinaloa. Therefore water hyacinth weevils(Neochetina eichhorniae Warner and N. bruchi Hustache), imported from the USA in1993, were used to initiate a biologicalcontrol program. Precautionary screeningrevealed that some were infected with amicrosporidian so disease-free colonies wereproduced by eliminating infected breedinglines. To demonstrate effectiveness prior toopen field releases, weevils were firstreleased in cages at field sites. Weevilintensity increased to 6.3 weevils/plant after320 days when the plants were all dead ordying. More than 8,600 N. bruchi and14,500 N. eichhorniae were then releasedat various sites during January 1995 to August1996. Waterhyacinth coverage declined atBatamote reservoir (134 ha) from 95% to <3%by 1997; at the 12-ha Hilda reservoir from100% in May 1995 to 1% by March 1998; at the42.3-ha Arroyo Prieto reservoir from 100% to1% during the same interval; and at theMariquita reservoir (492 ha), the largestreservoir in the Humaya system, from 394 ha(80%)to 98.4 ha (20%).     

Role of single-strand DNA 3′-5′ exonuclease ExoI and nuclease SbcCD in stationary-phase mutation in <Emphasis Type="Italic">Escherichia coli</Emphasis> K-12

In RecBCD⁺ cells, a mutated single-strand DNA 3′-5′ exonuclease ExoI (SbcB15) induced an increase in stationary-phase mutation. In sbcB15 cells, as in wild-type cells, these mutations partially required RecA, RecB, RecF, and expression of the LexA regulon. The absence of nuclease SbcCD in sbcB15 cells decreased stationary-phase mutation and induced an increase in the number of cell filaments. The absence of ExoI (Δxon) in wild-type or sbcC cells did not change significantly the stationary-phase mutation. Differences between the sbcB15 and ΔxonA cells suggest a correlation between level of SOS induction and the generation of stationary-phase mutations.     

In Rhodobacter sphaeroides, chemotactic operon 1 regulates rotation of the flagellar system 2

Rhodobacter sphaeroides is able to assemble two different flagella, the subpolar flagellum (Fla1) and the polar flagella (Fla2). In this work, we report the swimming behavior of R. sphaeroides Fla2(+) cells lacking each of the proteins encoded by chemotactic operon 1. A model proposing how these proteins control Fla2 rotation is presented.