Microbiology Research - Academic Journals

al., 1998; Heubuelt 1929) have already been described.
This article describes the cultural and the biochemical
characteristics of a new strain of bacteria and the result
of a phenotypic and phylogenetic analysis based on 16S
rRNA gene sequences.
MATERIALS AND METHODS
Isolation procedures
Isolation
The strain was isolated from soil in a potato field in Hamadan (Lat.
34˚47′46″ N, Long. 48˚30′57″ E), Iran (March 2004, GenBank
accession No. AY569007). Five hundred mg soil was shrilled in a
300 ml flask according to Drews (1968). The cells were grown at
28°C for 2 weeks. Then the cell suspension was inoculated into
agar media (basic mineral medium and 18 g agar-agar added to 1
liter of water) (Merck, Germany). Various types of colonies were
isolated and grown separately on agar plates with nitrite and
mineral salt (Merck, Germany) by multiple subcultures.
Culture conditions
The culture media was prepared as described by Drews (1968)
and Bock et al. (1990). The basic mineral was supplemented with
400 mg sodium acetate, 1500 mg yeast extract (Difco, USA) and
1500 mg peptone (Merck, Germany) in 1 L of water at pH 7.6 (for
aerobic-growth), and nitrate instead of peptone, as an electron
acceptor for anaerobic conditions. Master plates (Stock cultures)
were prepared under mixotrophic conditions.
Batch cultivation
Batch cultures were grown in 50 ml liquid media. Based mineral
medium (Merck, Germany) supplemented with sodium acetate,
yeast extract, and peptone for routine and mixotrophic cultivation
under aerobic conditions was used (Bock et al., 1990; Drews,
1968). For heterotrophic growth (Bock et al., 1990), the medium
was modified as follows: 1000 mg nitrate (Merck, Germany) as an
electron acceptor was added to 1 L of medium under anaerobic
conditions. All experiments were done at pH 6.7, 7.6, and 8.3 and
repeated at least 3 times.
Analytical procedures
Protein purification
The protein was purified from enzyme extracts and measured
exactly as previously described by Bradford (1976), Spector (1978),
Davie (1982) and Laemmli (1970). Membranes and nitrite
oxidoreductase were isolated and purified according to
Sundermeyer-Klinger et al. (1984).
Gel electrophoresis
SDS-PAGE (Merck, Germany) was performed as described by
Milde and Bock (1984) and Sundermeyer-Klinger et al. (1984). The
cytochrome spectra of cell-free extract of the new strain (104) was
determined as previously explained by Sorokin et al. (1998). A
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diode-array spectrophotometer (Hewlett Packard, USA) was used
for the investigation. Proteins were identified from polyacrylamide
gels by the method of Francis and Becker (1984).
DNA and 16S rRNA analysis
For isolation of DNA, 2 g of wet weight cells were suspended in 5
ml TE buffer (50 mmol Tris, 20 mmol EDTA, pH 8.0) (Merck,
Germany). Cell lysates were prepared as described by Kraft and
Bock (1984). Total DNA was isolated and purified according to
Marmur (1961). The G+C content was calculated from the
denaturizing rate according to De Ley (1970).
PCR amplification for the nearly complete 16S rRNA gene and
sequencing were done as described by Brinkhoff and Muyzer
(1997) and Muyzer et al. (1995). Sequences were compared using
ARB software (Ludwig et al., 2004). The 16S rRNA gene
sequences of the isolates were automatically aligned to sequences
stored in the ARB database.
Electron microscopy
Cells cultured under mixotrophic conditions were concentrated 100fold,
and methods for fixation, embedding, and ultra-thin sections
were those described by Bock and Heinrich (1969). Sections were
stained with uranyl acetate and lead citrate (Electron Microscopy
Sciences, USA). Electron micrographs were taken with a
transmission electron microscope (Carl Zeiss EM-900; Zeiss,
Germany) at 80 kV accelerating voltage. Negatives were scanned
at 1200 dpi resolution, by CanoScan 8800F (Canon, Japan), and
pictures were processed using Adobe ® Photoshop ® software (CS4
Extended, Middle East Version 11.0).
Phylogenic analysis
In order to establish the precise taxonomic position of unknown
bacterium, the entire 16S rRNA sequences of the strain (104) was
determined.
RESULTS
G+C analysis
The G+C content of the new strain 104 DNA was 59%.
This value is different from Nitrobacter winogradskyi (61.7
mol%), Nitrobacter hamburgensis (61.2-61.6 mol%),
Nitrobacter vulgaris (58.9-59.9 mol%) and Nitrobacter
alkalicus (61.5-62.4 mol%) (Bock et al., 1990; Sorokin et
al., 1998).
The derived 16S rRNA consisted of 1421 nucleotides.
The determined sequences were compared with those of
other 16S rRNA sequences available in the GenBank.
Nitrobacteria hamadaniensis (strain 104) with 95.9%
genetic homology with Caulobacter, and 96.3% with
Brevundimonas (Table 1). It has 86-87.4% genetic
homology to the genus Nitrobacter, which are classified
as one of the main classes of Caulobacteriaceae. A
phylogenic tree, depicting the relationship of unknown
bacterium with Caulobacteriaceae and close relatives,are
shown in Figure 1, and the sequence similarities are