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International Journal of Systematic and Evolutionary Microbiology (2007), 57, 1872–1875
Rheinheimera chironomi sp. nov., isolated from achironomid (Diptera; Chironomidae) egg mass
Malka Halpern,1 Yigal Senderovich1 and Sagi Snir2
1Department of Biology, Faculty of Science and Science Education, University of Haifa, Oranim,
2Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel
A Gram-negative, rod-shaped bacterial strain, designated K19414T, was isolated from achironomid (Diptera; Chironomidae) egg mass which was sampled from Kishon River in northernIsrael. Phylogenetic analysis based on the 16S rRNA gene sequence positioned the novelstrain among the genus Rheinheimera, with closest similarity to Rheinheimera pacifica KMM1406T. The levels of similarity to type strains of Rheinheimera species were lower than 96.5 %.
Isolate K19414T is aerobic, motile by means of a single polar flagellum, catalase-negative andoxidase-positive; growth was observed at salinities of 0–2 % NaCl and the temperature for growthranged from 4 to 40 6
C. The major cellular fatty acids are 16 : 0 (14.8 %) and 16 : 1v7cand/or 15 : 0 iso 2-OH (25.76 %). The DNA G+
C content is 49.9 mol%. On the basis of itsphenotypic properties and phylogenetic distinctiveness, strain K19414T (5LMG 23818T 5DSM18694T) was classified in the genus Rheinheimera as the type strain of a novel species, forwhich the name Rheinheimera chironomi sp. nov. is proposed.
The genus Rheinheimera was created by Brettar et al. (2002)
study was recently characterized as the type strain of
while screening blue-coloured bacterial isolates from differ-
Oceanobacillus chironomi (Raats & Halpern, 2007).
ent depth stations in the central Baltic Sea. At present, the
Strain K19414T is a motile Gram-negative rod, psychro-
genus comprises three species: Rheinheimera baltica (Brettar
tolerant and strictly aerobic. Its exact taxonomic position
et al., 2002), Rheinheimera pacifica (Romanenko et al.,
was determined by means of a polyphasic approach that
2003) and Rheinheimera perlucida (Brettar et al., 2006). All
included phenotypic properties and phylogenetic analysis
three species were isolated from marine environments. In
based on the 16S rRNA gene sequence.
this study, a new member of the genus Rheinheimera thatwas isolated from a freshwater insect egg mass is proposed
For electron microscopy, bacteria in LB agar medium were
suspended in saline. The samples were adhered to a carbon-coated grid and stained with 2 % uranyl acetate and photo-
Strain K19414T was isolated from a chironomid (non-
graphed under a JEM-1200EX electron microscope (JEOL).
biting midge) egg mass while the diversity of the culturable
Electron microscopy showed that the cells were rods with
bacteria in chironomid egg masses was under study(Halpern et al., 2007). Chironomid egg masses were
a polar flagellum (0.3–0.76
1.0–2.4 mm) (Supplementary
sampled in September 2004, as described previously
(Halpern et al., 2007). Egg masses were washed thoroughly
The 16S rRNA gene was analysed to determine its phylo-
with sterile saline water and then their homogenates were
genetic position. Universal bacterial primers 8f and 1512r
diluted and cultured directly on bacteriological media
(Felske et al., 1997) were used to amplify internal fragments
(Halpern et al., 2007). Strain K19414T was isolated from an
of the 16S rRNA gene. The amplified PCR product was
egg mass that was sampled from Kishon River in northern
purified with the Wizard PCR product purification kit
Israel, cultured on LB agar and incubated at 30 uC for 48 h
(Promega). Purified PCR products were sequenced with
in the dark. Another isolate from the above-described
primers 8f, 534r, 968f and 1512r as described in detail byRaats & Halpern (2007). This resulted in a sequence of
Abbreviations: ML, maximum likelihood; MP, maximum parsimony; NJ,
For identification of closest relatives, the 16S rRNA gene of
The GenBank/EMBL/DDBJ accession number for the 16S rRNA genesequence of strain K19414T is DQ298025.
strain K19414T was compared with those of previouslyreported strains available in EMBL (http://www.ebi.ac.uk/).
An electron micrograph of cells of strain K19414T and details of its fattyacid profile in comparison with those of related type strains are available
Using the BLAST program (version 2.0), the closest se-
as supplementary material with the online version of this paper.
quences obtained were those of R. baltica OS140 (96.9 %),
R. baltica OSBAC5 (96.7 %), R. pacifica KMM 1406T
profiles of fatty acid methyl esters. The major fatty acid
(96.5 %), R. perlucida BA131T (96.0 %), R. baltica OSBAC1T
components (exceeding 10 %) of strain K19414T are 16 : 0
(95.8 %), Alishewanella fetalis CCUG 30811T (95.8 %),
(14.80 %) and summed feature 3 (16 : 1v7c and/or 15 : 0 iso
Brenneria salicis DSM 30166T (91.5 %) and Colwellia piezo-
2-OH; 25.76 %) (Supplementary Table S1).
phila Y223GT (89.0 %). The sequences were aligned by the
For determination of the DNA G+C content, genomic
multiple alignment package CLUSTAL W. Phylogenetic trees
DNA of strain K19414T was prepared according to a
were generated by neighbour joining (NJ), maximum
modification of the procedure of Wilson (1987). The DNA
parsimony (MP) and maximum likelihood (ML). NJ and
G+C content was determined using HPLC analysis of
MP trees were obtained using the MEGA software package(Kumar et al., 2004), while ML was computed by
hydrolysed DNA according to Mesbah et al. (1989). The
(Felsenstein, 1993). For NJ, distance correction was per-
analysis was performed by the BCCM/LMG Bacteria
formed using the Kimura two-parameter model (K2ST)
Collection Identification Service (Laboratory of Micro-
using rate variation across sites. The bootstrap values
biology, Ghent University, Belgium). The G+C content of
obtained were from 1000 iterations. The NJ tree was drawn
by the MEGA software (Fig. 1). In contrast to the highest
The morphological, physiological and biochemical traits of
sequence similarity to R. baltica OS140 as found by BLAST,
strain K19414T are summarized in the species description
all phylogenetic inference methods located the novel strain
and in Table 1. API ZYM tests of strain K19414T revealed a
as a sibling to R. pacifica KMM 1406T.
broad set of 12 enzyme activities (Table 1). Phylogenetic
For phenotypic characterization, LB agar was used as the
relationships between strain K19414T and some related
basal medium with a final concentration of 0.5 % NaCl.
taxa are shown in Fig. 1. Comparative 16S rRNA gene
Salt tolerance was determined in LB agar containing varying
sequence analysis showed that the new isolate was phylo-
concentrations of NaCl at 37 uC. Growth at various tem-
genetically most closely related to Rheinheimera species,
peratures (4, 6, 8, 10, 15, 18, 25, 30, 32, 35, 37, 39, 40, 41
with 95.8–96.5 % sequence similarity to the type strains.
and 43 uC) was measured on LB agar. Growth under anaer-
The new isolate shared its main characteristics with
obic conditions was determined after incubation in an
Rheinheimera species, such as being aerobic and oxidase-
anaerobic chamber on LB agar supplemented with glucose
positive, motile rods by means of polar flagella, assimilat-
or nitrate. Strain K19414T had optimal growth between 0.5
ing N-acetylglucosamine and hydrolysing gelatin. Its
and 1 % NaCl and at 30–37 uC (Table 1).
DNA G+C content was close to those of the otherdescribed Rheinheimera species (48.9–49.9 mol%) (Table 1).
Biochemical tests were performed by means of API
Unsaturated fatty acids formed a major fraction of the
20E, API 20NE and API ZYM identification systems
total fatty acids in strain K19414T, as in the other described
(bioMe´rieux) at 37 uC. Carbon assimilation was analysed
strains of the genus Rheinheimera (Supplementary Table S1).
using Biolog GN microwell plates according to the manu-
Together with the findings mentioned above, strain K19414T
facturer’s instructions (release 3.50, version DE; Biolog).
showed significant characteristics that allowed clear differ-
The plates were incubated for 48 h at 37 uC. Wells that
entiation from all three described Rheinheimera species. It
changed to purple were marked as positive for metabolic
lacked catalase activity, grew at relatively low NaCl con-
activity. Sensitivity of the strain to antibiotics was tested
centrations (0–2 %), grew at 40 uC, which is the highest
by means of LB agar and Sensi-discs (BBL). Tests were
growth temperature described for species of this genus, and
was positive for malate assimilation and for valine arylami-
For the cellular fatty acid analysis, cells were cultured on a
dase, cystine arylamidase and a-mannosidase (Table 1). On
tryptic soy agar (Difco) at 28 uC for 24 h and then the fatty
the basis of phenotypic characterization and phylogenetic
acids were extracted. The fatty acid profile was analysed by
analysis (Stackebrandt & Goebel, 1994), we propose that
means of the MIDI/Hewlett Packard Microbial Identifi-
strain K19414T should be classified as the type strain of a
cation system (Analytical Services Inc.), which uses GC
novel species, Rheinheimera chironomi sp. nov.
Fig. 1. Unrooted NJ tree (MEGA3, based onalignment from CLUSTAL W) of strain K19414Tand its closest relatives from the genusRheinheimera and related taxa based on 16SrRNA gene sequences. Bootstrap values areindicated along branches. Branches found byML with P values ,0.01 are marked by anasterisk. Branches marked by a plus sign are inconsensus among all reconstruction methods.
Bar, 0.01 accumulated changes per nucleotideposition.
Table 1. Differential properties of strain K19414T and Rheinheimera type strains
Strains: 1, strain K19414T; 2, R. perlucida BA131T (data from Brettar et al., 2006); 3, R. baltica OSBAC1T(Brettar et al., 2002); 4, R. pacifica KMM 1406T (Romanenko et al., 2003). +, Positive; W, weakly positive;2, negative; ND, no data available.
*R. baltica OS550 grew in the presence of 6 % NaCl (Brettar et al., 2002).
International Journal of Systematic and Evolutionary Microbiology 57
Description of Rheinheimera chironomi sp. nov.
monomethyl ester, L-alanine, L-alanyl glycine, glycyl L-aspartic acid and glycyl
Rheinheimera chironomi [chi.ro9no.mi. N.L. gen. n. chiro-
strates; all other Biolog GN2 substrates are not utilized. The
nomi of Chironomus, named after the non-biting midge
G+C content of the type strain is 49.9 mol%.
insect from the genus Chironomus (Diptera; Chirono-midae) from which the type strain was isolated].
The type strain is strain K19414T (5DSM 18694T 5LMG
Cells are aerobic, Gram-negative, non-pigmented rods,
23818T), which is of freshwater origin.
1.0–2.4 mm long and 0.3–0.7 mm wide, occurring as singlecells or in pairs, and they are motile by means of a single
polar flagellum. Colonies are circular, non-pigmented,smooth and convex. Oxidase-positive, catalase-negative
Brettar, I., Christen, R. & Ho¨fle, M. G. (2002). Rheinheimera baltica
and able to reduce nitrate to nitrite. Sodium ions are not
gen. nov., sp. nov., a blue-coloured bacterium isolated from the
required for growth. Growth is observed in 0–2 % (w/v)
central Baltic Sea. Int J Syst Evol Microbiol 52, 1851–1857.
NaCl, but not in 3 % NaCl. Grows at 4–40 uC, but not at
Brettar, I., Christen, R. & Ho¨fle, M. G. (2006). Rheinheimera perlucida
41 uC. Hydrolyses gelatin and casein but not DNA. Grows
sp. nov., a marine bacterium of the Gammaproteobacteria isolated
on LB and half-strength marine agar. Does not haemolyse
from surface water of the central Baltic Sea. Int J Syst Evol Microbiol56, 2177–2183.
bovine blood and does not grow on MacConkey agar. Themajor measurable fatty acid components (exceeding 5 %)
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version3.5c. Distributed by the author. Department of Genome Sciences,
of the type strain are 11 : 0 3-OH (5.53 %), 12 : 0 3-OH
University of Washington, Seattle, USA.
(8.54 %) 15 : 0 (6.86 %), 16 : 0 (14.80 %), summed feature 3
Felske, A., Rheims, H., Wolterink, A., Stackebrandt, E. & Akkermans,
(16 : 1v7c and/or 15 : 0 iso 2-OH; 25.76 %), 17 : 0 (5.76 %),
A. D. L. (1997). Ribosome analysis reveals prominent activity of an
17 : 1v8c (7.56 %) and 18 : 1v7c (6.68 %). The following
uncultured member of the class Actinobacteria in grassland soils.
fatty acids are detected in the type strain as minor
components: 9 : 0 (0.2 %), 10 : 0 (1.2 %), 11 : 0 (1.35 %),
Halpern, M., Landsberg, O., Raats, D. & Rosenberg, E. (2007).
10 : 0 3-OH (0.53 %), unknown ECL 11.799 (2.63 %), 12 : 0
Culturable and VBNC Vibrio cholerae: interactions with chironomid
(1.06 %), 13 : 0 (0.62 %), 12 : 0 iso 3-OH (0.41 %), 14 : 0 iso
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(0.19 %), 14 : 0 (0.89 %), summed feature 1 (one or more
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: integrated software
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for molecular evolutionary genetics analysis and sequence alignment.
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Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise
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