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African Journal of Microbiology Research Vol. 6(24), pp. 5126-5133, 28 June, 2012Available online at http://www.academicjournals.org/AJMRDOI: 10.5897/AJMR11.1008ISSN 1996-0808 2012 Academic Journals

Full Length Research Paper

Introducing a novel facultative nitrifying bacterium,"Nitrobacteria hamadaniensis"

Mohammad Zare1, Mohammad Hassan Heidari2*, Farkhondeh Pouresmaeili3, Maryam Niyyati4

and Mohammad Moradi5

1Department of Plant Pathology, Faculty of Agriculture, University of Bu-Ali-Sina, Hamadan, Iran.

2Cellular and Molecular Biology Research Center, Faculty of Medicine, Shahid Beheshti University of Medical Sciences,

Tehran, Iran.3Department of Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

4Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical

Sciences, Tehran, Iran.5

Department of Microbiology, Faculty of Medicine, Kerman University of Medical Sciences, Kerman, Iran.

Accepted 5 April, 2012

A new nitrifying bacterium has been identified as "Nitrobacteria hamadaniensis",from a potato farm inHamadan, Iran. Its morphological and molecular characteristics were examined by electron microscopy,protein purification, SDS-PAGE and 16S rRNA analysis. It was cultured at the different conditions todetermine the optimum pH and the generation time. The cells are rod shaped, 0.3-0.40.8-1.2 m insize, and contains polar caps of intracytoplasmic membrane. The strain is lithotrophic and grow slowerthan heterotrophic strains. The best growth was observed at mixotrophic conditions. It grew at pHrange between 6.7 to 8.3 with an optimum pH at 7.6. Based on the growth conditions, the generationtime ranged from 7-16 h. The G+C content of this strain was 59 mol%. Also, 16S rRNA gene sequenceanalysis indicated that the bacterium represents a hitherto unknown line peripherally associated to theCaulobacteriaceae with low G+C relatives. The sequence of nearly complete 16S rRNA gene of the

strain is recorded in the GenBank under number AY569007. According to the phylogenetic analysis andphenotypic criteria, it is proposed that the bacterium should be assigned to a new genus Nitrobacteria.

Key words: Nitrobacter, nitrobacteria, nitrification, genotype, morphovar, biovar.

INTRODUCTION

Nitrifying organisms of the genus Nitrobacter arepolymorphic; which are mostly rods to pear shaped andpossess polar caps of cytomembranes. The major sourceof energy and reducing power is from the oxidation ofnitrite to nitrate. Some Nitrobacter strains are able to

growth in heterotrophic conditions with acetate (Smithand Hoare, 1968), or pyruvate (Bock, 1976) as carbonsource. These organisms are also facultative (Bock et al.,1988; Freitag et al., 1987). Nitrite-oxidizing bacteria areubiquitous in terrestrial and aquatic natural environmentsunder moderate conditions (Bock and Koops, 1992;

*Corresponding author. E-mail: [email protected]. Tel:+982123872584. Fax: +9821 22171928.

Laanbroek and Woldendorp, 1995; Both et al., 1992)There are some indications that nitrifying bacteria mayalso be present in extreme environments such as acidsoils (De Boer and Laanbroek, 1989; De Boer et al.1991; Hakinson and Schmidt, 1988), and acid sulfinic ore

(Bock et al.,1992). These bacteria have been found inalkaline environments such as saline soda lakes andsoda soil samples in Wadi Natrun, Egypt (Imhoff et al.1979). The nitrite oxidoreductase consisted of threemajor proteins with apparent molecular weights of116.000, 65.000 and 32.000 kDa (Sundermeyer-Klingeet al., 1984). These species of Nitrobacter, that isNitrobacter winogradskyi (Watson et al., 1981Winogradsky, 1892; Engel et al., 1954), Nitrobactehamburgensis (Bock et al., 1983), Nitrobacter vulgaris(Bock et al., 1990), and Nitrobacter alkalicus (Sorokin et
http://www.academicjournals.org/AJMRhttp://www.academicjournals.org/AJMR


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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 resultof a phenotypic and phylogenetic analysis based on 16SrRNA gene sequences.

MATERIALS AND METHODS

Isolation procedures

Isolation

The strain was isolated from soil in a potato field in Hamadan (Lat.344746 N, Long. 483057 E), Iran (March 2004, GenBankaccession No. AY569007). Five hundred mg soil was shrilled in a300 ml flask according to Drews (1968). The cells were grown at28C for 2 weeks. Then the cell suspension was inoculated intoagar media (basic mineral medium and 18 g agar-agar added to 1liter of water) (Merck, Germany). Various types of colonies wereisolated and grown separately on agar plates with nitrite andmineral 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 with400 mg sodium acetate, 1500 mg yeast extract (Difco, USA) and1500 mg peptone (Merck, Germany) in 1 L of water at pH 7.6 (foraerobic-growth), and nitrate instead of peptone, as an electronacceptor for anaerobic conditions. Master plates (Stock cultures)were prepared under mixotrophic conditions.

Batch cultivation

Batch cultures were grown in 50 ml liquid media. Based mineralmedium (Merck, Germany) supplemented with sodium acetate,yeast extract, and peptone for routine and mixotrophic cultivationunder aerobic conditions was used (Bock et al., 1990; Drews,1968). For heterotrophic growth (Bock et al., 1990), the mediumwas modified as follows: 1000 mg nitrate (Merck, Germany) as anelectron acceptor was added to 1 L of medium under anaerobicconditions. All experiments were done at pH 6.7, 7.6, and 8.3 andrepeated 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 nitriteoxidoreductase were isolated and purified according toSundermeyer-Klinger et al. (1984).

Gel electrophoresis

SDS-PAGE (Merck, Germany) was performed as described byMilde and Bock (1984) and Sundermeyer-Klinger et al. (1984). Thecytochrome spectra of cell-free extract of the new strain (104) wasdetermined as previously explained by Sorokin et al. (1998). A

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diode-array spectrophotometer (Hewlett Packard, USA) was usedfor the investigation. Proteins were identified from polyacrylamidegels 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 5ml TE buffer (50 mmol Tris, 20 mmol EDTA, pH 8.0) (MerckGermany). Cell lysates were prepared as described by Kraft andBock (1984). Total DNA was isolated and purified according toMarmur (1961). The G+C content was calculated from thedenaturizing rate according to De Ley (1970).

PCR amplification for the nearly complete 16S rRNA gene andsequencing were done as described by Brinkhoff and Muyze(1997) and Muyzer et al. (1995). Sequences were compared usingARB software (Ludwig et al., 2004). The 16S rRNA genesequences of the isolates were automatically aligned to sequencesstored in the ARB database.

Electron microscopy

Cells cultured under mixotrophic conditions were concentrated 100fold, and methods for fixation, embedding, and ultra-thin sectionswere those described by Bock and Heinrich (1969). Sections werestained with uranyl acetate and lead citrate (Electron MicroscopySciences, USA). Electron micrographs were taken with atransmission electron microscope (Carl Zeiss EM-900; ZeissGermany) at 80 kV accelerating voltage. Negatives were scannedat 1200 dpi resolution, by CanoScan 8800F (Canon, Japan), andpictures were processed using Adobe

Photoshop

software (CS4

Extended, Middle East Version 11.0).

Phylogenic analysis

In order to establish the precise taxonomic position of unknownbacterium, 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(61.7mol%), Nitrobacter hamburgensis (61.2-61.6 mol%)Nitrobacter vulgaris (58.9-59.9 mol%) and Nitrobactealkalicus (61.5-62.4 mol%) (Bock et al., 1990; Sorokin eal., 1998).

The derived 16S rRNA consisted of 1421 nucleotidesThe determined sequences were compared with those oother 16S rRNA sequences available in the GenBankNitrobacteria hamadaniensis (strain 104) with 95.9%genetic homology with Caulobacter, and 96.3% withBrevundimonas (Table 1). It has 86-87.4% genetichomology to the genus Nitrobacter, which are classifiedas one of the main classes of Caulobacteriaceae. Aphylogenic tree, depicting the relationship of unknownbacterium with Caulobacteriaceae and close relatives,areshown in Figure 1, and the sequence similarities are


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Table 1. Similarity matrix of 16S rRNA sequences.

1 2 3 4 5 6 7 8 9 10 11 12 13

Afipia clevenlandensisAfipia felis 98.6Blastobacter denifricans 96.5 96.5Bradyrhizobium japonicum 97.9 97.3 98.2Rhodopseudomonas palustris 96.9 96 97.3 98.3Nitrobacter winogradskyiATTCC 25381 97.4 96.9 97.1 98.2 97.1Nitrobacter winogradskyiATTCC 14123 97 96.7 96.5 97.9 96.9 98.7Nitrobactersp. strain R6 96.8 96.4 96.7 97.7 96.7 98.8 99.3Nitrobacter hamburgensis strain x 14 96.6 96.3 96.8 97.7 97.5 98 98 98Nitrobacter hamburgensis strain nb 14 96.9 96.5 96.9 97.9 97.2 98.4 98.3 98.4 99.5Nitrobacter alkalicus strain AN1 97.3 96.9 97.2 98.3 97.2 99.1 99.2 99.2 98.4 98.6Nitrobacter alkalicus strain AN2 97.4 97.5 97.1 98.3 97.2 99 99.1 99.1 98.3 98.6 99.9Nitrobacteria hamadaniensis strain 104 86.3 85.3 86 86.3 86.6 86.2 87.1 86.7 86.1 86.7 87.4 87.5Brevundimonas diminuta 87.7 87.1 87.8 87.8 87.8 87.7 87.1 87.6 88 87.6 87.8 87.7 96.3

Figure 1. A phylogenetic tree derived from 16S rRNA gene sequences, the tree was created by using the neighbor-joining method and

values, showing the phylogenetic interrelationships between Nitrobacteria hamadaniensis and other close relatives. The bootstrap valuesindicated.


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Table 2. Influence of organic compounds on growth of the nitrite-oxidizing strain 104 underanaerobic conditions at different pH values in batch culture

a.

Growth condition Strain 104 growth Amounts of NO2 concentration (mmol)

pH 8.3 Weak 0.15

pH 7.6 Weak 0.16

pH 6.7 Weak 0.1aSpecific activity of the strain 104 was weak in heterotrophic condition at different pH values.

indicated in Table 1. The comparisons help distinguishthe new bacterium located at the periphery of theCaulobacteriaceae.

Protein analysis

The protein analysis of cell free extracts of the strain

(104) grown at pH 7.6, indicated -bands (437 and 589nm in size), presenting nitrite cytochrome spectracomparable to those of other Nitrobacter species. Thisstrongly suggests that the new strain belongs to theNitrobacteria genus. In addition to -bands, cytochrome c550 and cytochrome c oxidase type 3 (maximum 550nm up to 607 nm) were other prominent features of thestrain, respectively.

Physiological characteristics

Nitrobacteria hamadaniensis grew optimally at 26-28Cand pH 7.6. Colonies on agar plates formed within 3, 4and 12 d under mixotrophic, heterotrophic, andlithotrophic conditions. Colonies on mineral salt agarplates sized 0.1 mm in diameter, were orange, circular,and swelled. Optimum growth rates were obtained inmixotrophic medium containing nitrite, sodium acetate,yeast extract and peptone.

Strain 104 was able to grow under nitrite-oxidizinglithoautotrophic, mixotrophic and heterotrophic conditionsat pH 6.7 to 8.3. The stoichiometry analysis, conversionof nitrite to nitrate in batch culture was 96.3-99.1% andnitrate to nitrite, under anaerobic condition was 1-1.6%(Table 2). The organisms grew on mineral mediumsupplemented with organic compounds such as sodium

acetate, yeast extract and peptone as sources of energyand carbon. Batch cultivation at different pH valuesclearly demonstrated that the nitrite-oxidizing strain (104)isolated from soil belonged to facultative neutrophilicspecies. It could grow within a pH range of 6.7 to 8.3(Figure 4). The growth rate at pH below and above 7.6,was extremely slow. During growth at pH above 7, cellsstarted to branch. Their optimum growth was close totheir upper pH limit (around 7.6). The main difference ofthe strain 104 from all four known species of the genus

Nitrobacter (Nitrobacter winogradskyi, Nitrobactehamburgensis, Nitrobacter vulgaris and Nitrobactealkalicus) was the rapid growth on culture medium usedfor cultivation of strain (104) with a starting pH 7.6 and anitrite concentration of about 1 g. Strain 104 was able togrow in nitrite limited culture media within a broad pHrange from 6.7 to 8.3 with an optimum pH 7.6 (Figure 4)The doubling times of autotrophically and mixotrophically

grown Nitrobacteria strain 104 was 16 and 7 h at pH 7.6respectively. This was higher than the rate described foneutrophilic species grown lithoautotrophicaly with nitrite(Bock and Koops, 1992; Keen and Prosser, 1987). Oustudy shows that organic compounds had an influence onthe growth of nitrite-oxidizing strain 104 from soil, at pH6.7 to 8.3, during 5 d incubation. There were nosignificant differences between the bacteria activity inheterotrophic with 1000 mgP/L nitrate under anaerobicconditions at different pH values in batch culture (Table2).

The pH profile in the kinetics of oxidation in batchculture was significantly different for the cells grown atdifferent pH values. The profile for the rate of nitriteoxidation (Figure 4) measured with cells grown at pH 6.7was similar to that measured for Nitrobacter species(Hunik et al., 1993). The curve had its maximum at pH7.6 and decreased at a pH higher than 8. The nitrite-oxidizing activity measured with cells grown at pH 7.66.7, and 8.3 that was maximal at pH 7.6, respectivelyand incubation time was 192 h.

SDS-PAGE analysis

The results of SDS-PAGE of cell-free extracts, based onphenotypic criteria, showed that Nitrobacteria

hamadaniensis is composed of 4 bands, 2 strong andprominent bands of ~116 kDa, one 67 kDa, and a 14 kDaband, respectively (Figure 3).

DISCUSSION

A new species of bacteria was identified, that wasdifferent from Nitrobacter winogradskyi (Bock 1976)Nitrobacter hamburgensis (Bock et al.,1983), and Nitro-


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a b

Figure 2. Electron photograph ofNitrobacteria hamadaniensis ultrasturcture. Thin section from

grown cell mixotrophically and harvested during exponential phase of growth, showing lamellarmembrane system, poly--hydroxybutyrate (PHB), and polyphosphate granules (X: 50,000).PHB = Poly--hydroxybutyrate. PP = Polyphosphate granules. CM = Cytoplasmic membrane.

Figure 3. SDS-PAGE (12.5% acryl amide) of

cell free extract from Nitrobacteriahamadaniensis protein stained withcomassie blue R- 250.

bacter vulgaris (Bock et al., 1990). The new bacteriagrew at all the different culture conditions, as reported bySteinmueller and Bock (1977), and the growth rates inmixotrophic media could be used for taxonomicpurposes. Our observations showed that this neworganism, like Nitrobacter vulgaris (Bock et al., 1990)grows by dissimilation and reduces nitrate where nitrateis present as an alternative electron acceptor. The cellsofNitrobacteria hamadaniensis had a similar shape, size,and ultrastructure. This is further evidence to prove theexistence of the new Nitrobacteria species. The newisolate is different from most of the other Gram negative

bacteria. They are short rod cells, pear shaped, 0.3-0.40.8-1.2 m in size, and motile. They tend to form

flocks (Figure 2) and/or biofilms on the glass surface ofculture flasks. Cell division normally occurres by budding.

The cytoplasmic membrane protrudes into thecytoplasm, forming a polar cap of interacytoplasmicmembranes. Carboxysomes were found in the strainsgrown under chemolithotrophic conditions, but this wasnot observed under mixotrophic conditions. The othetypical inclusion bodies were poly--hydroxybutyrate andpolyphosphate granules. These results correspond to theother four strains, Nitrobacter winogradskyi(Winogradsky, 1892), Nitrobacter hamburgensis (Bock eal., 1983), Nitrobacter vulgaris (Bock et al., 1990), andNitrobacter alkalicus (Sorokin et al., 1998). However, in

the new strain the upper band was clearly separated.The phenotypic criteria are similar to the recent findings

obtained by Samelis et al. (1995). This suggests thaeach species yields a specific protein profile identical tothe respective type strain. Bock et al. (1990) reported thathere are significant differences between protein profilesof Nitrobacter winogradskyi, Nitrobacter hamburgensisNitrobacter vulgaris and Nitrobacter alkalicus.

In conclusion, the new nitrite-oxidizing bacteria strain104 isolated from soil differs from previously describedspecies by their potential to grow and oxidize nitrite at pH7.6. The batch continuous cultivation showed theiremarkable ability to adapt to abroad range of pH values

Our data shows that the analysis of the whole celprotein was a quick and effective method to distinguishany bacteria among Nitrobacteria hamadaniensis.

Species description

Description of Nitrobacteria hamadaniensis sp. nov.

nitro bacteria. npl (NL. fr. Nitr- + bacteria): the soibacteria concerned in nitrification. Nitrobacteria


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Zare et al. 5131

NO2

NO2

Figure 4. A-B. Influence of pH and culture condition on dynamic of nitr ite oxidation of Nitrobacteria hamadaniensis strain 104 in

Batch culture and influence of pH on oxidation activity of washed cells grown in Batch culture at pH 6.7-8.3. The culture started towash out at D = 0.008 h

-1. A. Lithoautotrophic condition. B. Mixotrophic condition. C-D. Influence of pH on the growth of

Nitrobacteria hamadaniensis strain 104 in Batch culture with 14.5 mmol nitrite at pH 6.7, 7.6 and 8.3 (Culture wash out at D = 0.008h

-1). C. Nitrite oxidizing activity of cell cultivated at lithoautotrophic with nitrite. D. Nitrite oxidizing activity of cell cultivated at

mixotrophic condition.

hamadaniensis = hamadani, ensis. Hamadan Iranian

place name; M.L. adj. hamadaniensis of Hamadan. Thecells are Gram negative, short rods to pear shaped with asize of 0.3-0.40.8-1.2 m. Each cell contains severalcarboxysomes and posses a polar cap ofcytomembranes to form flattened vesicles. Cells produceextracellular polymers at all growth conditions causingthe formation of a biofilm. Facultative lithoautotrophsoxidize nitrite to nitrate under aerobic conditions andreduce nitrate to nitrite under anaerobic conditions. Cellsgrow chemolithotrophically, heterotrophically, or

mixotrophically. The growth rate in chemo-organic

medium is more rapid than in chemolithotrophic mediumThe surfaces of colonies on mineral salt agar plates are0.1 mm in diameter after 12 d at 28C and pH valuesbetween 7.6-7.8. They have two prominent proteins o116 and 67 kDa, and a 14 kDa protein which appears asa faint band. The G+C content of DNA is 59 mol% andthe sequence of nearly complete 16S rRNA gene ostrain 104 is stored in the GenBank database and Japancollection of microorganisms under accession numbers

AY569007(http://www.ncbi.nlm.nih.gov/Genbank/index.ht


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ml), and JCM 14789 (http://jcm.riken.go.jp/JCM/catalogue.shtml), respectively. Nitrobacteriahamadaniensis is deposited in Persian type culturecollection under the number, PTCC 1681(http://www.irost.org/en/ptcc/index.asp?code=1#).

ACKNOWLEDGEMENTS

We thank M. Piriai and F. Niazi (Department of Anatomy,Faculty of Medicine, Shahid Beheshti University ofMedical Sciences) for their excellent technical assistance.

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