Seasonal distribution and sex ratio of Autographa gamma (L.) and Trichoplusia orichalcea (Fabricius) (Lep., Noctuidae) from São Miguel (Azores)

A dinâmica do estado adulto de Autographa gamma (L.) e Trichoplusia orichalcea (Fabricius) (Lepidoptera, Noctuidae) foi estudada entre Julho de 1988 e Dezembro de 1989, através de armadilhas luminosas do tipo pensilvânia instaladas em três localidades da ilha de São Miguel (Ribeira Grande, Arribanas e Lagoa do Congro). Para as três localidades, foram evidenciadas flutuações de densidade consideráveis. A. gamma foi observada continuamente nas três localidades, enquanto T. orichalcea foi capturada durante todo o ano na Ribeira Grande, em Fevereiro e entre Setembro e Novembro na Lagoa do Congro e entre Setembro e Dezembro nas Arribanas. Para as duas espécies, ambos os sexos foram capturados simultaneamente. O sex-ratio foi favorável às fêmeas, exceptuando para a localidade das Arribanas, onde os machos de T. orichalcea foram mais frequentes do que as fêmeas.ABSTRACT: The adult dynamics of Autographa gamma (L.) and Trichoplusia orichalcea (Fabricius) (Lepidoptera, Noctuidae) were studied between July of 1988 and December of 1989, at Ribeira Grande, Arribanas and Lagoa do Congro on the island of São Miguel, using Pennsylvania backlight traps. Despite evidence for considerable density fluctuations, A. gamma was continuously present at the three locations. In contrast T. orichalcea was only captured the whole year at Ribeira Grande, during February and from September to November at Lagoa do Congro, and from September to December at Arribanas. For any given species both sexes were captured simultaneously. In T. orichalcea males were more frequent than females only at Arribanas while at the other localities females of this species and of A. gamma were more abundant

Construction of the <i>X. citri ssuA</i>-deleted mutant (<i>Xac::ssuA</i>) and complemented strain (<i>Xac::ssuAc</i>).

<p>(A) Chromosomal deletion of the <i>ssuA</i> gene was obtained after electroporation of the suicide pNssuA plasmid into the <i>X. citri</i> 306 strain. (I) The first step in the construction of the <i>X. citri</i> mutant was the insertion of a 2-kb fragment encoding resistance to spectinomycin and streptomycin into the <i>KpnI</i> site of the <i>ssuA</i> gene, originating within the pNssuA (8,152 bp) vector. (II) After transformation of wild-type <i>X. citri</i> with pNssuA, a double recombination event generated the <i>Xac::ssuA</i> mutant strain (III), which was screened by selection of cells resistant to both spectinomycin and sucrose. (B) PCR amplification of <i>ssuA</i> genes of selected <i>X. citri</i> colonies using primers FssuA2Nde28a and RssuA2Hind28a. Samples: P, molecular weight markers; 1–3, colonies selected for resistance to spectinomycin and sucrose. The presence of a single 2-kb band indicates a successful gene replacement event (samples 1 and 2), while amplification of two bands of 1 kb and 2 kb in size indicates the presence of the chromosomal wild-type gene and a copy of the mutated <i>ssuA</i> gene (sample 3). (C) Strategy of cloning and digestion analysis of the pKX33-p<i>ssuA</i> plasmid. The localization of <i>ssuA</i> gene and the promoter region in the <i>ssu</i> operon are evidenced in yellow and red colours, respectively. A band of 1,926 bp was generated after cleavage of the pKX33-p<i>ssuA</i> plasmid with <i>Sal</i>I and <i>Xba</i>I restriction enzymes.</p

The presence of SsuA in <i>Xanthomonas</i> species and the genetic organisation of the s<i>su</i> operon in <i>E. coli</i> and <i>X. citri</i>.

<p>(A) Neighbour-joining tree based on 16S rRNA processing protein RimM showing relationships among <i>Xanthomonas</i> species and other species encoding SsuA proteins (black balls). Distances were determined using sequences aligned by ClustalW. (B) Genetic organisation of the <i>ssu</i> operon in <i>E. coli, X. citri</i> and other <i>Xanthomonas</i> species in which it was found. The amino acid sequence identities of the orthologues related to <i>X. citri</i> proteins are indicated as percentages inside the arrows. Genes are represented by the same colours used for the <i>X. citri</i> operon. SsuA: periplasmic-binding protein; SsuB: nucleotide-binding protein; SsuC: ABC transporter permease; SsuD: NAD(P)H-dependent FMN reductase; SsuE: alkanesulphonate monooxygenase FMNH(2)-dependent.</p

Crystallisation, X-ray diffraction pattern and determination of the tertiary structure of <i>X. citri</i> SsuA.

<p>(A) SsuA crystals grown in 0.1 M HEPES, pH 7.3, 1.5 M ammonium sulphate and 0.1 NaCl using 6 mg/ml of protein in 20 mM Tris buffer, pH 7.0, containing 50 mM NaCl. (B) Diffraction pattern of SsuA crystal at 2.0 Å resolution. Data were collected at the D03B-MX1 beam line Brazilian Synchrotron Light Laboratory (LNLS) using 1.433 Å radiation and recorded on a <i>MARCCD</i>165 detector (oscillation data with Δφ = 1.0^o). (C) Cartoon illustration of the overall structure of SsuA bound to HEPES, MOPS and MES (stick) showing the alpha-beta structures of domains I (deep blue and cyan) and II (orange and yellow). The N-terminus is shown in domain I.</p

Lack of SsuA affects <i>in vitro</i> growth and xanthan gum production by <i>X. citri</i>.

<p>Growth curve of <i>X. citri</i> wild type (A) and the <i>Xac::ssuA</i> mutant (B) in M9 media supplemented with sulphate or different alkanesulphonate sources. Samples were taken every 2 h for measuring the growth of the samples. (C) The <i>Xac::ssuA</i> mutant shows altered colony morphology after growth at 30°C in LB plates. The strain recovered the normal colony morphology after complementation with the <i>ssuA</i> gene. (D) Production of xanthan gum by the parental and <i>ssuA</i> mutant strain after 24 h of growth in LB broth. Complementation with pKX33-p<i>ssuA</i> restored the reduced xantham gum production observed in the <i>Xac::ssuA</i> mutant.</p

Ligand-binding site and interactions of the <i>X. citri</i> SsuA protein.

<p>(A) Ligand interactions of SsuA and HEPES, MES and MOPS. Domains I and II are coloured in blue and yellow, respectively; the residues involved in the ligand interaction, as well as the ligands themselves, are shown as sticks. (B) SsuA topology and conservation of the ligand-binding sites in different orthologues. The residues that form the pocket and those that interact with ligands are marked in clear and dark grey, respectively. The residues marked in green are unique to <i>X. citri</i> SsuA. The numbering follows the <i>X. citri</i> SsuA sequence. Xac: <i>X. citri</i> (GI: 21243924); Avi: <i>Azotobacter vinelandii</i> (GI: 67153714); Spr: <i>Serratia proteomaculans</i> (GI: 5604713); Eco: <i>E. coli</i> (GI: 90111189); Sfl: <i>Shigella flexneri</i> (GI: 161486517); Ype: <i>Yersinia pestis</i> (GI: 22124165); Rso: <i>Ralstonia solanaraceum</i> (GI: 207723295); Ppu: <i>Pseudomonas putida</i> (GI: 167031279); Bph: <i>Burkholderia phytofirmans</i> (GI: 187921640); and Atu: <i>Agrobacterium tumefaciens</i> (GI: 159184964). (C) Structure of SsuA in ribbon diagram showing the positioning of the two helices and the beta-sheet (forest cartoon) that form the dipole. The ligand-binding pocket and HEPES inside are shown, respectively, in transparent surface and stick. (D) Electrostatic potential at the surface of the two helices (α2 and α5) and the beta-strand (β3) that contain the residues for dipole formation and sulphate group coordination. (E) Structural superposition of secondary structures of periplasmic-binding proteins (grey) with dipoles similar to that found in the SsuA structure (shown in red). The superposed helices and β-strands belong to the <i>E. coli</i> aliphatic sulphonate-binding protein (PDB 2X26), <i>Sinechocystis</i> sp. 6856 nitrate-binding protein (PDB 2G29), <i>E. coli</i> phosphate-binding protein (PDB 1IXH), <i>Thermus thermophilus</i> glutamate/glutamine-binding protein (PDB 1US5) and <i>X. citri</i> molybdate-binding protein (PDB 2H5Y).</p