Effects of flooding and temperature on Aedes albifasciatus development time and larval density in two rain pools at Buenos Aires University City

Aedes albifasciatus is a floodwater mosquito that breeds in temporary waters. This semi-domestic species, widely distributed in Argentina, is a competent vector of the western equine encephalitis. The present study was carried out in two rain pools of the city of Buenos Aires, from April 1998 through March 1999. Samples were taken twice a week during the cold season and daily during the warmer months, starting from October. Immature mosquitoes were collected with a dipper, being the number of dippers proportional to the flooded area. The estimated rainfall thresholds to initiate cohorts of Ae. albifasciatus were: 16-17 mm in the fall-winter period, 25 mm in the spring, and 30 mm in the summer. The development time of the different cohorts and the mean air temperature of their respective periods were estimated in all seasons, ranging from six days (at 24 degress C) to 32 days (at 13 degrees C). The equation that best expresses the relationship between development time and mean air temperature is dt =166,27.e(-0,1435.T) (R(2)=0,92). Significantly shorter development times were recorded for larvae of the first three stages as compared to the fourth larval stage and pupae.     

RAPD variation in relation to population size and plant fitness in the rare Gentianella germanica (Gentianaceae)

We investigated the distribution of genetic variation and the relationship between population size and genetic variation in the rare plant Gentianella germanica using RAPD (random amplified polymorphic DNA) profiles. Plants for the analysis were grown from seeds sampled from 72 parent plants in 11 G. germanica populations of different size (40-5000 fruiting individuals). In large populations, seeds were sampled from parents in two spatially distinct subpopulations comparable in area to the total area covered by small populations. Analysis of molecular variance revealed significant genetic variation among populations (P <0.001), while genetic variation among subpopulations was marginally significant (P <0.06). Average molecular variance within subpopulations in large populations did not differ significantly from whole-population values. There was a positive correlation between genetic variation and population size (P <0.01). Genetic variation was also positively correlated with the number of seeds per plant in the field (P <0.02) and the number of flowers per planted seed in a common garden experiment (P <0.051). We conclude that gene flow among natural populations is very limited and that reduced plant fitness in small populations of G. germanica most likely has genetic causes. Management should aim to increase the size of small populations to minimize further loss of genetic variation. Because a large proportion of genetic variation is among populations, even small populations are worth preserving.     

Stereo- and Regioselective Hydroxylation of α-Ionone by Streptomyces Strains

A total of 215 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 15 strains showed little conversion to 4-hydroxy- and none showed conversion to the 3-hydroxy product as desired. Among these 15 Streptomyces strains, S. fradiae Tü 27, S. arenae Tü 495, S. griseus ATCC 13273, S. violaceoniger Tü 38, and S. antibioticus Tü 4 and Tü 46 converted α-ionone to 3-hydroxy-α-ionone with significantly higher hydroxylation activity compared to that of β-ionone. Hydroxylation of racemic α-ionone [(6R)-(−)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity.     

Bradyrhizobium japonicum FixK2, a Crucial Distributor in the FixLJ-Dependent Regulatory Cascade for Control of Genes Inducible by Low Oxygen Levels

Bradyrhizobium japonicum possesses a second fixK-like gene, fixK₂, in addition to the previously identified fixK₁ gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates fixK₂, whose product then activates fixK₁. While the target genes for control by FixK₁ are unknown, there is evidence for activation of the fixNOQP, fixGHIS, and rpoN₁ genes and some heme biosynthesis and nitrate respiration genes by FixK₂. FixK₂ also regulates its own structural gene, directly or indirectly, in a negative way.     

Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.The antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] (PRN) is produced by many pseudomonads and has broad-spectrum antifungal activity (1, 5, 12–14, 17). PRN has been implicated as an important mechanism of biological control of fungal plant pathogens by several Pseudomonas strains (12–14), including P. fluorescens BL915, from which the prn genes were isolated (10).Tryptophan was identified as the precursor for PRN, based on the feeding of cultures with isotopically labeled and substituted tryptophan (2, 7, 8, 17, 25). Biosynthetic pathways were proposed as early as 1967 (7) and have been refined on the basis of tracer studies and the isolation of intermediates (Fig. ​(Fig.1)1) (2, 8, 17, 19, 23, 25). Recently, Hammer et al. (9) described the cloning and characterization of a 5.8-kb DNA region which encodes the PRN biosynthetic pathway. This DNA region confers the ability to produce PRN when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for PRN production. In the research described here, we used mutants in which each of the four genes was disrupted and strains which overexpress the individual genes to elucidate the function of each gene product in PRN biosynthesis. Open in a separate windowFIG. 1Biosynthetic pathways for PRN as proposed by van Pée et al. (23) (A) and by Chang et al. (2) (B). The reactions catalyzed by the PRN biosynthetic enzymes encoded by the prnABCD genes are indicated above the appropriate reaction arrows.

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are described in Table ​Table1.1. Pseudomonas strains were cultured in Luria-Bertani medium at 28°C. Antibiotics, when used, were added at the following concentrations: tetracycline, 30 μg/ml; and kanamycin, 50 μg/ml. The expression vector pPEH14 consists of the P_tac promoter and rrnB ribosomal terminator from pKK223-3 (Pharmacia, Uppsala, Sweden) cloned into the BglII site of the broad-host-range plasmid pRK290 (4). P_tac is a strong constitutive promoter in Pseudomonas (unpublished data). The PRN biosynthetic genes are the coding regions described by Hammer et al. (9). Each coding region was cloned from the translation initiation codon to the stop codon by PCR with restriction sites added to the ends to facilitate cloning. For prnB, the native GTG initiation codon was changed to ATG. The clones were sequenced after PCR.

TABLE 1

Bacterial strains and plasmids used in this study

Generation and Properties of a Streptococcus pneumoniae Mutant Which Does Not Require Choline or Analogs for Growth

A mutant (JY2190) of Streptococcus pneumoniae Rx1 which had acquired the ability to grow in the absence of choline and analogs was isolated. Lipoteichoic acid (LTA) and wall teichoic acid (TA) isolated from the mutant were free of phosphocholine and other phosphorylated amino alcohols. Both polymers showed an unaltered chain structure and, in the case of LTA, an unchanged glycolipid anchor. The cell wall composition was also not altered except that, due to the lack of phosphocholine, the phosphate content of cell walls was half that of the parent strain. Isolated cell walls of the mutant were resistant to hydrolysis by pneumococcal autolysin (N-acetylmuramyl-l-alanine amidase) but were cleaved by the muramidases CPL and cellosyl. The lack of active autolysin in the mutant cells became apparent by impaired cell separation at the end of cell division and by resistance against stationary-phase and penicillin-induced lysis. As a result of the absence of choline in the LTA, pneumococcal surface protein A (PspA) was no longer retained on the cytoplasmic membrane. During growth in the presence of choline, which was incorporated as phosphocholine into LTA and TA, the mutant cells separated normally, did not release PspA, and became penicillin sensitive. However, even under these conditions, they did not lyse in the stationary phase, and they showed poor reactivity with antibody to phosphocholine and an increased release of C-polysaccharide from the cell. In contrast to ethanolamine-grown parent cells (A. Tomasz, Proc. Natl. Acad. Sci. USA 59:86–93, 1968), the choline-free mutant cells retained the capability to undergo genetic transformation but, compared to Rx1, with lower frequency and at an earlier stage of growth. The properties of the mutant could be transferred to the parent strain by DNA of the mutant.Pneumococci differ from other gram-positive bacteria in that their lipoteichoic acid (LTA) and wall teichoic acid (TA) have the same chain structure which is, moreover, unusually complex (Fig. ​(Fig.1):1): glycerophosphate is replaced by ribitol phosphate (7), and between the ribitol phosphate residues a tetrasaccharide is intercalated (23). It contains d-glucose, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (AATGal), and two N-acetyl-d-galactosaminyl residues, one or both of which carry a phosphocholine residue at O-6 (references 3 and 12 and this report). Open in a separate windowFIG. 1Pneumococcal TA and LTA. As shown, in strain R6 most of the repeats carry two phosphocholine residues each, at O-6 of the N-acetyl-d-galactosaminyl residues (3, 12). In strain Rx1 and Rx1/AL⁻, most repeats contain one phosphocholine residue (this report) attached to O-6 of the non-ribitol-linked galactosaminyl residue (14).Pneumococci are not able to synthesize the choline required for the synthesis of these substituents. Moreover, choline is an essential growth factor (2, 30) but can be substituted in this function by nutritional ethanolamine (EA) (38). Phosphoethanolamine is incorporated into LTA and TA in place of phosphocholine (14), but it cannot replace phosphocholine functionally. Phosphocholine-substituted LTA serves to anchor pneumococcal surface protein A (PspA) to the outer layer of the cytoplasmic membrane, with choline-mediated interaction between membrane-associated LTA and the C-terminal repeat region of PspA. In EA-grown bacteria, PspA is no longer retained and is released into the surrounding medium (45). Phosphocholine substituents also play an essential role for the activity of the major pneumococcal autolysin, an N-acetylmuramyl-l-alanine amidase (38). This protein possesses a choline-binding C-terminal domain that is essential for activity but, unlike PspA, is not essential for retention on the pneumococcal cell surface (16, 32). Binding of phosphocholine-substituted LTA to this domain results in potent inhibition of the amidase (21). The inhibitory property is dependent on the micellar structure of LTA (13) and lost by deacylation (5). Phosphocholine-substituted LTA may also participate in the transport of the amidase through the cytoplasmic membrane from the cytosol (5), the location of its synthesis (15). It additionally effects the conversion of the inactive E form of the enzyme into the active C form (5). This conversion is likewise effected by the choline residues of cell wall-linked TA (33, 39). Furthermore, binding of the amidase to the choline residues of TA is prerequisite for the hydrolysis of cell walls by the enzyme (18, 22). It should be noted that the amidase is not essential for growth. Though the enzyme is completely inactive in EA grown cells, the growth rate is not affected. However, cell separation is impaired, and there is a loss of stationary-phase and penicillin-induced cell lysis (38, 40), as well as a loss of genetic transformation (38). After insertional inactivation of the autolysin gene (lytA), the autolysin-deficient mutants (Lyt⁻) grew normally (31) and did not even show impeded cell separation (41).In this report, we describe a mutant which acquired the ability to grow in the absence of choline and analogs. Except for the observation that [³H]choline-substituted LTA is not a precursor of [³H]choline-substituted TA (6), nothing is known about the biosyntheses of pneumococcal LTA and TA and the stage of biosynthesis at which phosphocholine is incorporated. Since the absence of choline incorporation might affect the structure of LTA and TA as well as the composition of cell walls, we included relevant analyses in our study.(A preliminary report of this work was presented in an overview on pneumococcal LTA and TA at the International Meeting on the Molecular Biology of Streptococcus pneumoniae and Its Diseases, Oeiras, Portugal, September 24 to 29, 1996 [10].)