SALT INDUCIBLE-PROTEINS AND CONJUGAL GENE TRANSFER OF HALOTOLERANT Staphylococcus ISOLATED FROM SALINITY SOIL

alotolerent bacteria are those that can tolerate a broad range of NaCl concentrations (0-32% w/v) (Hezayen et al., 2010). So, there are different categories of halotolerant microbes: not tolerate, those which tolerate only a small concentrations of salt about 1%, slightly tolerant (6-8%), moderately tolerant (1822%) and extremely tolerant, those microbes that grow over the whole range of salt concentrations (0-32%) (Larsen, 1986). Recently, Parthiban et al. (2010) classified halophilic bacteria according to their salt requirement and growth pattern to slight halophiles which growth at 2-5% NaCl, moderate halophiles which growth at 5-20% NaCl and extreme halophiles which growth at 20-30% NaCl. Extreme halophiles are microorganisms that grow under hostile to most organisms. Some of them, such as bacteria which thrive in hypersaline environments have been recognized for their use in biotechnological remediation applications. The applied of halophilic bacteria include recovery of saline soil by directly supporting of growth of vegetation thus indirectly increasing crop yields in saline soil. The other application of halophilic bacteria was in food and pharmaceutical industries, production of enzymes, polymers and various cosmetic products. The possibility of application of halophilic bacteria in soil is recovery and the importance of microbial diversity in soil (Kannika, 2003).


compatible
solutes.Hence these microorganisms are able to adapt to a wide range of salt concentrations.The compatible solutes include polyols such as glycerol, sugars and their derivatives, amino acids and their derivatives as well as quaternary amines such as glycine betaine and ectoines.Compatible solutes display a general stabilizing effect by preventing the unfolding and denaturation of proteins caused by heating, freezing and drying (Ventosa et al., 1998).The salt-in strategy is employed by true halophilies, including halophilic archaea and extremely halophilic bacteria.These microorganisms are adapted to high salt concentrations and cannot survive when the salinity of the medium is lowered.
They generally do not synthesize organic solutes to maintain the osmotic equilibrium.This adaptation involves the selective influx of K + ions into the cytoplasm.All enzymes and structural cell components must to be adapted to high salt concentrations for proper cell function (Shivanand and Mugeraya, 2011).
Extreme environments such as acidic, thermophilic, hypersaline, and arid regions, are important 'hot spots' of microbial 'megadiversity'.These are habitats of microorganisms which have the genetic and physiological capacity to survive and grow under these harsh or extreme conditions (Woese, 1987;Olsen et al., 1994).Extensive studies have been made in recent years into hyper saline environments resulting in a large number of new halophilic species being isolated e.g.Oceanobacillus aswanensis, it was isolated from salted fish sauce in Aswan city, Egypt (Hezayen et al., 2010), Paenibacillus chungwensis which isolated from Marakanam salterns in India (Parthiban et al., 2010).Gram-negative halophilic (Vibrio, Alteromonas, Acinetobacter, Marinomonas and Pseudomonas) (Prado et al., 1991) and Gram positive halophilic (Staphylococcus, Marinococcus, Sporosarcina Salinococcus and Bacillus) have been recovered from saline soils, salterns and activated sludge (Farrow et al., 1992;Ajibola et al., 2005;Olukanni et al., 2006;Elisangela et al., 2009).Staphylococcus spp., Micrococcus spp.and Bacillus spp.have been isolated from sea water and tropical marine fish but little information has been reported on the species level identities or specific sources of these bacteria (Surendran et al., 1989;Uddin et al., 2001;Rao and Surendran, 2003;Swaminathan et al., 2007;Jeyasekaran et al., 2008).
The main objective of this study was to (1) isolation and characterization of some salt tolerant bacteria from salinity soil in Sharkia Governorate, (2) determine the protein pattern in halophilic bacteria and (3) studying the ability of salt tolerant gene(s) to transfer by natural gene transfer mechanisms.

MATERIALS AND METHODS
This study was carried out in Microbial Genetics Lab., Genetics Dept.and Bacterial Lab., Agriculture Microbiology Dept., Faculty of Agriculture, Zagazig University, Egypt.

Soil samples
Soil samples were collected from salinity soils of Sharkia Governorate (El-Hessenia and Abo-Kibeer).

Saline solution
The saline solution consisted of 9 g/per liter NaCl (Miller, 1992).

Antibiotics
All the antibiotics were used in the current study are commercially and purchased from pharmaceuticals.The tetracycline and septazoal were prepared with concentration from 50-500 μg/ml while the streptomycin, ampcillin, chloramphenicol and penicillin were prepared with concentration from 500-2000 μg/ml

Acridin orange
A ten mg/ml concentration of acridin orange was used according to Guha et al. (1997).

Phosphate buffer
Phosphate buffer was prepared from 1/15 M potassium phosphate and 1/15 M disodium phosphate.

Bacterial isolation and identification
About 25 g of the soil samples was added to 225 ml of sterile saline solution in sterile flask.The flasks were placed on magnetic stirrer for 15min then serial dilutions were made.A 0.1ml from the above dilutions was spreaded on SM agar plates and incubated at 30ºC for 72 h.A colony of each morphology on SM plates was selected for purification and identification.Identification was carried out according to morphological and biochemical characters according to Krieg and Holt (1984)  The isolates were inoculated in LB medium and incubated overnight at 30C, 1 ml from each isolate was inoculated in 50 ml SM with different concentrations of NaCl,11.7,17.6,23.4,29.3 and 35.1% NaCl (w/v)).After interval time (1.0, 2.0, 4.0, 7.0 and 10.0 days), serial dilutions were made to determine the total viable counts of the isolates (Moukhtar et al., 2005).Control was carried out using LB medium.

Sensitivity of Staphylococcus spp. isolates to some antibiotics
Different concentrations of six common antibiotics were used.Loop of liquid culture of each isolate was streaked onto surface of SM agar plates containing antibiotic and the control without antibiotics.All the plates were incubated at 30C for 72 h.

Lysogenicity test of Staphylococcus spp. isolates
Each strain of Staphylococcus (1.0 ml) was inoculated into 50 ml of LB medium, and then placed on a shaker incubator for overnight at 30C.UV induced agent was used at 5, 10 and 15 min.Centrifugation was carried out at 10,000 rpm for 15 min of UV treated and non-treated bacterial suspension.The supernatant was removed and passed through a sterile membrane (0.45 μm) and spotted onto layer of bacterial cells (Howaida et al., 2007).

Plasmid curing of Staphylococcus spp. isolates
To determine the location of salt tolerant gene (s), plasmid curing was carried out by acridin orange.The isolates were grown overnight in 5 ml LB medium.Next day, 0.2 ml of cultures were added to 5 ml SM liquid medium and incubated for overnight.A 0.2 ml of the previous cultures was added to 2 ml of LB medium containing 10 mg/ml acridin orange.All the tubes were incubated in the dark at 30C for overnight.A 0.1 ml from the above tubes were spread over LB agar plates and incubated at 30ºC for 24 h.A total of 70 colonies were selected and replicated onto LB and SM agar by replica plate technique (Chin et al., 2005;Ajaz et al., 2009;El-Deeb, 2009).

SDS-PAGE of Staphylococcus spp. isolates proteins
Total cellular protein was extracted as described by Laemmli (1970) and subjected to SDS-polyacrylmide gel electrophoresis.
The isolates were inoculated in LB medium and incubated overnight at 30ºC.A 1 ml from each isolate was inoculated into 50 ml SM with different concentrations of NaCl (11.7,17.6,23.4,29.3 and 35.1%), the control was LB medium.All cultures were incubated at 30C for 24 h.At the end of experiment, bacterial cells were separated from all treatments by centrifugation at 13400 rpm for 10 min then dispersed in saline solution (8.5 g NaCl/L).An aliquot of 50 μL of the bacterial suspension was combined with 50 μL of the sample buffer (1 M Tris-HCl, 50% glycerol, 10% SDS, 10% β-mercaptoethanol, 0.1% Bromophenol blue, pH 6.8) and heated at 100C for 10 min then loaded in 12% SDS-PAGE.After electrophoresis, the gel was stained in 50 ml of staining solution for 30-45 min with shaking at room temperature and distained in distaining solution.The gel was dried for 20 h and photographed.The photographs were analyzed using Gel Analyzer (Egygene) Software.

Bacterial conjugation
The salt tolerant Staphylococcus isolates were used as donors (streptomycin resistant and ampecillin sensitive).The non-tolerant Micrococcus sp. was used as recipient (streptomycin sensitive and ampecillin resistant).Overnight cultures of donors and recipient (0.2: 0.2 ml) were mixed and spread onto LB agar plates.
The plates were incubated at 30C for overnight; the mixtures were collected and re-suspended in phosphate buffer.Serial dilutions were made and plated onto selective media for donor (SM agar and str), recipient (LB agar and amp) and transconjugants (SM agar and str and amp).

Quantitative and qualitative assessment of the isolates
Isolation was performed on SM agar.In total 100 isolates out of 60 (60%) of the Gram-positive bacteria were identified in this study and the other 40 isolates (40%) was gram negative bacteria.The gram-positive isolates were distributed in 7 genera: Staphylococcus, Micrococcus, Bacillus, Corynebacterium, Streptococcus, Enterococcus and Sporosarcina.Fifty percent of the Grampositive bacteria were not identifiable because bacterial growth was insufficient to allow identification.The Gram-negative isolates were distributed in 3 genera: Pseudomonas, Enterococcus and Escherichia.The gram negative isolates were not identifiable to species levels.

Speciation and diversity of Gram-positive bacteria in salinity soil
Using the Staphylococcus API testand Biolog Microlog1 4.20 system at Cairo Mircen, 20 of the isolates were confirmed to be Staphylococcus spp.Seven of 20 Staphylococcus isolates were identified to species level with 5.0 isolate identified as S. xylosus, two as S. kloosii, and 5.0 as S. arlettea, one as S. equorum, four as S. saprophyticus, two as S. gallinarum and one as S. succinus.Staphylococcus species accounted 66.67% of the identified Gram-positive bacteria.Staphylococcus arlettea, S. saprophyticus and S. xylosus were the most frequent species (70% prevalence) representing 25%, 20% and 25% of Staphylococcus species respectively.Staphylococcus species are known as halotolerant bacteria which habitat in different saline environments such as salted fish (Rifky, 1993), seawater (Kakizaki et al., 2008), activated sludge (Elisangela et al., 2009), vegetable pickled plants (Abou-Elela et al., 2010) and from marine fish (Al Bulushi et al., 2010).In total, five isolates were identified as Micrococcus by conventional biochemical tests.Micrococcus species accounted for 16.67% of the identified Gram-positive bacteria.Micrococcus luteus was the most dominant Micrococcus species.About five isolates of Bacillus bacteria were identified to genus level by conventional biochemical tests; these accounted for 16.67% of the identified Gram positive bacteria.

Effect of salinity on growth/survival rate of Staphylococcus species
To assess bacterial salt tolerance, the growth/survival behavior of the bacteria was analyzed in response to increasing salt concentration in.As shown in  (Shivanand and Mugeraya, 2011).The optimal growth was occurred at 11.7 to 17.6% NaCl for all Staphylococcus species after two days.S. xylosus, S. kloosii (1), S. arlettea and S. equorum are extreme halophiles which growth at 23.4% NaCl.However, other tested bacteria are moderately halophilic which growth at 11.7-17.6%NaCl).This result agreed with the described for moderately halophilic bacteria which suggests the requirement of salt at concentration ranging from 2.9% up to 14.6% NaCl in culture medium (Ventosa et al., 1998;Echigo et al., 2005).
According to Parthiban et al. (2010), the above isolates are moderate halophiles which growth at 5-20% NaCl concentrations.Some of them, such as bacteria which thrive in hyper-saline environments have been recognized for their use in biotechnological remediation applications (Kannika, 2003).

Biochemical genetic variation of cell protein content
Protein contents of S. xylosus, S. kloosii (1), S. arlettea, S. equorum, S. saprophyticus, S. gallinarum, S. succinus and S. kloosii (2) cells grown in free salt LB medium and SM medium with different concentrations of NaCl were compared by SDS electrophoretical analysis (Fig. 2  There was induction of some new proteins not present in the free salt medium and inhibition of production of some others proteins that were produced in the free salt medium.There was also an increase, as well as a decrease in the level of expression of some proteins.All these differences may be directly associated with the bacterial response of to salt stress.The induction of novel proteins or the increased production of already existing proteins, which are only produced under stress conditions, is responsible for stress responses.The decease in production or the inhibition of production of certain proteins is most probably the result of high levels of proteins modification or gene regulation, caused by a decrease in metabolic activity (Faiza et al., 2011).

Antibiotic resistant pattern
An antibiotic resistant is one of the most important criteria for persistence and competition of target bacteria with other bacteria in habitat.Furthermore, pattern of antibiotics is important for identification and scrimination of bacteria.As well as antibiotic resistance could be used as a selectable marker.So, the strains of Staphylococcus were tested for resistance of some of common antibiotics.Table (2) shows that, all Staphylococcus species were sensitive to ampicillin, chloroamphenicol and penicillin at all used concentrations (500-2000 μg/ml), except S. arlettea and S. equorum were resistant to chloroamphenicol at 500 μg/ml so, these 3 antibiotics are most efficiency against the tested strains.On the other hand, all strains were resistant to streptomycin at all concentrations except S. saprophyticus and S. kloosii (2) at 500 μg/ml only.S. xylosus, S. arlettea and S. equorum were resistant to septazol at all concentration and tetracycline at 50 or 100 μg/ml.
In the past, penicillin, erythromycin, clindamycin, and/or gentamicin were used for treating form staphylococcal infections.However, owing to many factors, including the extensive use of these antibiotics, staphylococci have developed resistant populations (Varaldo, 1989).The part of this development may be due to mutations and the other to transfer of plasmids between not only different strains or species (Dale, 1998) but also between genera (Hassan, 2010).It is early known that the plasmids carry a number of different antibiotic resistance genes (Dale, 1998).The resistance of tested strains to antibiotics indicated that these strains have one or more plasmid.The confirmed existence of plasmids in halophilic bacteria cell has been represented (Arvanitis et al., 1995, Moukhtar, 2005;Ghosh et al., 2010).

Lysogenecity test of Staphylococcus species
Lysogenicity test of halotolerant Staphylococcus species was carried out to detection of spontaneously released or UV induced bacteriophages (Table 3).No phage was detected, so no strain was lysogen under this current study.The failure of halotolerant Staphylococcus species to be lysogens may be due to the effect of salt on the relationship between phage and bacterial cell.Growing lysogenic cells in media containing NaCl concentrations progressively increased the amount of bacteriophage released.So the salt affects on lysogen stability by stimulating mutagenesis of the repressor gene.The salt stress alters the ability of phages to maintain the lysogenic state (Shkilnyj and Koudelka, 2007).These findings are consistent with the ideas that, an increased external NaCl increases Na + within the cell, this led to decreases lysogen stability by disrupting repressor DNA interactions.In contrast, other studies showed that successfully isolation of bacteriophages from halophilic bacteria obtained from soil (Calvo et al., 1988;Ventosa et al., 1998).

Plasmid curing of Staphylococcus species
To determine the location of salt tolerant gene(s), plasmid curing was carried out by acridin orange.It was suggested previously that all isolated strains must be contain plasmid(s) because it were resistant to one or more of antibiotics.Plasmid curing experiments were performed to recognize the role of plasmid in salinity resistance.After curing the strains were placed on SM medium, it found that all selected colonies (70) were tolerated to salt.These results indicated that plasmid(s) do not carry any salt tolerant genes in the used strains.So, the tolerant genes should be carried on chromosomal DNA.These results are in agreement with the results of Moukhtar (2005), it showed that salt tolerant genes are located on chromosomal DNA of Halococcus salifodinae and Bacillus pasteurii.

Bacterial conjugation
The salt tolerant Staphylococcus species were used as donors (streptomycin resistant and ampecilline sensitive).The non-tolerant Micrococcus sp. was used as recipient (streptomycin sensitive and ampecilline resistant).Table 4 shows that the number of transconjugants was ranged between 1.19 X 10 3 and 3.28 X 10 3 and conjugation frequency was ranged from 3.3 X 10 -7 to 9.1 X 10 -7 .The transconjugants cells were detected on selective media containing 11.7% NaCl salt concentrations, so, all colonies that could grow onto this media must be salt tolerant.These results indicated that the salt tolerant gene(s) were able to transfer by conjugation.Conjugation considered the only genetic transfer mechanism described for the bacterial halophiles.Natural transformation has not been reported and approaches such as electroportion or CaCl 2 treatment have either been unsuccessful or given no reproducible results (Ventosa et al., 1998).Although some bacteriophages have been described for halophiles, transduction methods have not yet been developed.A majority difficulty in the use of halophiles for genetic transfer experiments is that at their optimal salinity they generally tolerate high concentrations of most antimicrobial agents (Nieto et al., 1993).However, a decrease of the salinity resulted in an enhanced sensitivity of Halomonas, Chromohalobacter and Salinivibrio strains to many antimicrobials (Coronado et al., 1995).Transfer frequency was affected by many factors such as cell growth phase, mating time, donorto-recipient ratio and composition as well as salinity of the mating medium (Vargas et al., 1997).
In the future study, these strains may be used to detection and isolation of salt tolerant gene(s) and transfer it to plants.This may be attempting to solve problems of saline soil that reduced agriculture production in over the world.Both physical and chemical methods were not cost-effective for soil reclamation of the reservoir.

survival rate of Staphylococcus spp. Isolates under different salt concentrations.
and Table1).It was observed that, S. saprophyticus, S.