Random Selection Of Micro-Organisms ; 2) Hyphomicrobium :

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Hope you all are fine and enjoying the articles of Random Selection of Microorganisms on Concept Of Microbiology .

So today we are going to explore the Bacteria 

Called : Hyphomicrobium 

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Concept Of Microbiology 

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Study of Hyphomicrobium 

1) Hyphomicrobium :

Hyphomicrobium is a denitrifying methylotroph, meaning that they reduce nitrate, and use methane as carbon and energy source. It has been suggested that Hyphomicrobium can oxidize manganese, but whether they can harvest energy from this process is not known. In this study, soil samples are taken from different environments in Woods Hole and enriched for hyphomicrobium. I obtained enrichment cultures containing up to 90% hyphomicrobium and measured the rates of denitrification under different conditions. Manganese concentration had some effect on cell growth and respiration, but the mechanism is inconclusive at this level of resolution.

A Hyphomicrobium is a genus of Gram-negative, non-spore-forming, rod-shaped bacteria from the family of Hyphomicrobiaceae. It has a large polar or sub-polar filiform prostheca very similar to that of Caulobacter. In addition to having a nutritional function, the prostheca also plays a role in the initiation of DNA replication.

2)Scientific Taxonomy :

Kingdom  : Bacteria

Phylum    : Proteobacteria

Class.      : Alpha-Proteobacteria

Order       : Rhizobiales

Family     : Hyphomicrobiaceae

Genus     : Hyphomicrobium

Species  : chloromethanicum,Coagulan etc.

Introduction : 

i) Hyphomicrobium is a dimorphic α-Proteobacteria. They reproduce by budding and form two daughter cells that are morphologically and behaviorally different : one nonmotile prosthecate that has elongated cylindrical appendages, and the other becomes a motile flagellated swarmer cell.

ii) This morphological separation results in the spatial separation of the daughter cells to increase the efficiency in nutrient acquisition (Garrity et al., 2006; Moore, 1981). 

iii) Cell morphology varies with growth conditions. In media with low nutrient concentration, hyphae may elongate up to 300µm. The unique morphological characteristics facilitate the relatively easier recognition and identification of hyphomicrobium from environmental samples.

iv) Hyphomicrobium are metabolically variable. They are facultative anaerobes, meaning they can switch between oxygen and nitrate as electron acceptors. They can use a variety of carbon compound as both energy and carbon source, including methylamine, di- and trimethylamine, dichloromethane and methylsulfate (Garrity et al., 2006; McDonald et al., 2001). 

v) Methanotrophy is possible with low concentration of oxygen (Amaral & Knowles, 1995). Their capability of coupling methanol oxidation and denitrification is of special interest in waste water treatment, which removes nitrate from eutrophic environments at the expense of methanol(Green et al., 2010; Liessens et al., 1993). 

vi) The goal of this study is to enrich and isolate hyphomicrobium strains from different environments in Woods Hole, and examine its morphology and growth. 

vii) Another objective is to measure rate of denitrification of hyphomicrobium in culture, assessing it’s ecological importance in the nitrogen cycle. The third interest is to test whether some hyphomicrobium stains are capable of manganese oxidation and whether the energy is harvested to promote cell growth.

3) Ecology : 

i) Hyphomicrobium sp. are comomn in wastewater plants and very well discussed for their indicating denitrification. Hyphomicrobium are often noted because of their unique apperance and reproduction by budding. 

ii) You have a "mother", a non-motile cell with a cylindrical stalk protruding from the floc. The "daughter" cell that eventually breaks away from the mother is motile bacteria (by way of a bacterial flagella) that allows for the daughter cell to colonize new areas. 

iii)The stalk can be up to 3 um long so it can be observed with a phase contrast microscope under 400x magnification.

 

Fig.Budding Process you can observe above👆


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iv) So if you see an increase in Hyphomicrobium  in your system, you may have:

• More methanol or short-chain soluble organics
• Higher nitrate and/or lower D.O. (remember lower DO can exist inside the floc)

v) Overall Hyphomicrobium  are a common water bacteria that can increase in populations when conditions are sufficient. 

vi) If you are required to remove Nitrate/Nitrite for effluent permits, seeing Hyphomicrobium can be a very good indication of healthy denitrification.

4) Method and Materials :

i) Soil samples were collected from four different locations in Woods Hole: near School St. swamp (swamp), near fire hydrant by Stoney Beach (pond), under pine tree by garbage beach (GB), tarmac driveway by fire hydrant (tarmac). 

ii) A small scoop of soil was inoculated to a Pfenning bottle containing selective medium. The medium contains following components : 171 µM NaCl, 19.7 µM MgCl2, 6.8 µM CaCl2, 67.1 µM KCl, 20 mM pH 7.2 MOPS buffer, 1X HCl-dissolved traced elements, 200 µM Na2SO4, 100 µM pH 7.2 K3PO4, 5mM NH4Cl, 50mM KNO3, 2.5ml methanol, 1X multivitamin solution. Samples were incubated at 30 ˚ C. 

iii) Enrichments were achieved through series transfers of 2ml of culture into fresh medium. Enrichment medium was prepared anoxically and all procedures were performed inside anaerobic chamber. 

iv) Liquid cultures were streaked onto agar plates containing same components as the liquid medium after three transfers.

v) After three transfer, 1 ml of each of the four liquid cultures were transferred into four sets of selective medium containing different Mn2+ concentration : trace, 10µM, 50µM and 100µM Mn2+ .

vi) The liquid cultures were also streaked on selective agar plates with same series of Mn2+ concentrations.

vii) Subsamples were taken from each enrichment bottle from the Mn2+ matrix everyday after inoculation. Growth conditions were monitored by measuring optical density of the subsamples at 600 nm wavelength.

viii) 75 µL of subsamples were mixed with 25 µL ethanol to fix the cell. 1.5 µL of the mixture were added to 5 ml PBS and filtered onto 0.2 µL membrane filter. Filters were stained with DAPI for total cell count.

ix) Mixture were also analyzed using phase contrast microscope for relative abundance of hyphomicrobium in the culture based on morphology. Remaining subsamples were filtered through 0.2 µL syringe filter and stored at -20 °C for nitrate analysis. 

x)Nitrate concentrations were measured using NOx box in Wankel lab at WHOI as described (Braman & Hendrix, 1989; Garside, 1982

5) Pathogenicity 

• Methanol dehydrogenase (MDH) from Methylobacterium extorquens, Methylophilus methylotrophus, Paracoccus denitrificans and Hyphomicrobium X all contained a single atom of Ca2+ per alpha 2 beta 2 tetramer.
• 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties .
• In addition, subculture of this enrichment culture on potential intermediates in the degradation pathway of linuron (i.e., N,O-dimethylhydroxylamine and 3-chloroaniline) resulted in the isolation of, respectively, Hyphomicrobium sulfonivorans WDL6 and Comamonas testosteroni WDL7.
• Sequence analysis showed the presence of some organisms whose closest relatives are known iron- and manganese-oxidizing/reducing bacteria, including Hyphomicrobium, Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea .
• Dimethylsulfone as a growth substrate for novel methylotrophic species of Hyphomicrobium and Arthrobacter.

6) Antibiotic Resistance : 

• methicillin-resistant Staphylococcus aureus (MRSA)
• vancomycin-resistant Enterococcus (VRE)
• multi-drug-resistant Mycobacterium tuberculosis (MDR-TB)
• carbapenem-resistant Enterobacteriaceae (CRE) gut bacteria.

 Context of Hyphomicrobium :

• Chloromethane-dependent expression of the cmu gene cluster of Hyphomicrobium chloromethanicum .
• Timing of swarmer cell cycle morphogenesis and macromolecular synthesis by Hyphomicrobium neptunium in synchronous culture.
• CmuA from IMB-1 has high sequence homology to the methyltransferase CmuA from Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum and contains a C-terminal corrinoid-binding motif and an N-terminal methyltransferase motif .
• These results are consistent with the lack of an active pyruvate dehydrogenase complex which would make it impossible for Hyphomicrobium X to convert pyruvate into acetyl-CoA and to generate energy from carbon compounds for which the energy metabolism relies on oxidation through tricarboxylic acid (TCA) cycle intermediates.

7) Cell Structure :

So what does having Hyphomicrobium mean to operating a WWTP? Looking at Hyphomicrobium metabolism:

• ​Use methanol and other short chain soluble organic compounds as an energy source. Even if these compounds are not present in your influent, they are often formed as bacteria degrade higher molecular weight organics.
• Prefer to use oxygen for respriation but are also very capable of using nitrate and nitrite as an alternative electron acceptor.
• Potential - not fully documented - to grow on energy provided by manganese oxidation. (This is still being discussed)
• Mesophilic with individual species in the Hyhomicrobium genus having different nitrate reductases, salinity toleranes, and organic energy sources.
• Hyphomicrobium is a genus of Gram-negative, non-spore-forming, rod-shaped bacteria from the family of Hyphomicrobiaceae.^{[1][2][3][4][5][6]} It has a large polar or sub-polar filiform prostheca very similar to that of Caulobacter. In addition to having a nutritional function, the prostheca also plays a role in the initiation of DNA replication.
  
• Reproduction in Hyphomicrobium begins when its hyphal filament (prostheca) grows from one end of the cell (this happens when the cell no longer has a flagellum) and the bud grows from the tip of the prostheca that eventually differentiates into a new swarmer cell 

8) Metabolism : 

i) The yield of Hyphomicrobium EG on dimethyl sulphoxide, dimethyl sulphide and methylamine, considering the metabolic pathways of these compounds, suggested that the organism gained energy from the oxidation of the sulphur moiety of the former compounds. 

ii) Indeed, a comparison of chemostat cultures of Hyphomicrobium EG grown on methylamine in the presence and absence of sulphide or thiosulphate proved this obligate methylotroph to be a chemolithoheterotroph.

iii) Enzyme- and respiration data showed that the organism had constitutive enzymes for the breakdown of dimethyl sulphoxide although they were clearly regulated to need. 

iv) Addition of sulphide or thiosulphate to methylamine-limited chemostat cultures of Hyphomicrobium EG not only resulted in the induction of enzymes necessary for their breakdown, but also caused the enzymes for dimethyl sulphoxide metabolism, especially methyl mercaptan oxidase, to be induced.

v) The formation of H₂O₂, a product of the latter enzyme, was reflected in the relatively high catalase activities during growth on dimethyl sulphoxide and in the organisms inability to grow on this compound in the presence of a catalase inhibitor.

9) Resluts & Conclusions : 

Two days after transfer into the Mn2+ enrichment matrix, all cultures turned turbid. Bubbles rose in the liquid when loosening the bottle caps (Fig 1). 

After 72 hours, cell density in all enrichment exceeded 108 cells / ml. Relative abundance of hyphomicrobium were counted based on morphology using a light microscope (Fig. 2). 

The flagellate cells accounted for between 64.5% and 89.6% of the total.

Fig. 1 Nitrogen gas forming upon loosening the cap

Fig. 2 Wet mount of liquid culture used for examining relative abundance of hyphomicrobiumpopulation

 Enrichment from soil near garbage beach contained fastest growing cells and highest relative abundance of hyphomicrobium. 

Cell density of enrichment tarmac and pond were lower, but relative abundances of hyphomicrobium were comparable to the other two sets of enrichments. After 72 hours of incubation, nitrate in the enrichments were mostly depleted, thus not able to support continuous growth of hyphomicrobium. 

The plateaus in growth curves were probably due to lack of nitrate instead of stationary growth of the culture. In future enrichment, this problem could be avoided by increasing starting nitrate concentration, or spiking nitrate into medium during the incubation. 

Nitrates consumption occurred at a much higher rate than normally measured from the field. Considering the ubiquitous distribution of hyphomicrobium and high abundance in the environment, they can be important players in the biogeochemical cycling of nitrogen.