海洋微生物菌群分析

1、The Pseudomonas community in metal-contaminated sediments as revealed by quantitative PCR: a link with metal bioavailabilityReportor: Ma XiaoXuMembers:Ma XiaoXu(1225109)Wang ZiJian(1213525)Lu DeQing(1213419)Introduction for Pseudomonas1.Pseudomonas bacteria are ubiquitous Gram-negative and aerobic m

2、icroorganisms that are member of the -Proteobacteria.2.Pseudomonas bacteria are also responsible for different human infections and some species have developed resistance to antibiotics ，leading to the expansion of nosocomial diseases .3.Reasona.oxidative degradation processes and biosurfactant prod

3、uctionb.harbor metal resistance mechanisms such as efflux pumps and intracellular redox enzymes4.function bioremediation toolsDefine the concept of bioavailable regarding metals1.For a unicellular organism,a compound may be defined as bioavailable when it is able to cross the cellular envelope at a

4、given time . Once inside the cell, metals may have various effects according to their concentration and chemical properties.2.When concentration levels are too high, bacteria usually react by the expression of specific metal resistance systems such as P-type ATPases, metallothioneins, RND efflux pum

5、ps and/or CDF transporters.3.It is therefore expected that bacteria displaying the most efficient resistance systems are selected in metal-contaminated environments where metals are bioavailable.Aim to the experiment1.Use quantitative PCR with specific primers to estimate the biomass of the whole Ps

6、eudomonas community in 4 aquatic sediments.2.Total metal levels and bioavailable metals were estimated in the sediments using, respectively, strong acids and 1 M HCl treatment.3.Use cell cultivation to test the metal resistance capacities of some Pseudomonas isolates obtained at the most contaminate

7、d station.MaterialStation 130 is situated inthe North Sea, on the Belgian Continental Plate. It presentslow concentrations of metals in comparison to the otherfreshwater sitesThe MetalEurop site is the most metal contaminatedsite of the present study.The Raches site is mildlycontaminatedFerin is a c

8、ontrol site, supposedly uncontaminatedQuantification of metals1. Total metals (TE)To compare the total metal content at different sampling sites, a metal pollution index (MPI) was calculated with the following equation.2. Bioavailable metals (BM)metals were extracted from 1 g of wet sediment (anoxic

9、) using 1 M HCl for 24 h (in triplicates). Each sample was then filtrated through a 0.45 mm filter and metal concentrations were measured using inductively coupled plasma atomic emission spectroscopy.3. acid volatile sulfides(AVS)-to calculate a toxicity index (TI)wet sediments (1 g, anoxic) were mi

10、xed with 6 M HCl for 1 h at 25 C. The H2S gas that was produced was then trapped in a 1 M NaOH solution. The sulfide concentration was measured with a sulfide-specific electrode (Orion).Quantification of Pseudomonas by quantitative PCRDNA extractionA260/A280 quality and measure the concentrationPseu

11、domonas lipoprotein oprI gene, Pseudomonas 16S rRNA gene, bacterial 16S rRNA gene Standard curveIsolation of metal-tolerant bacteria and strain characterization1.Enrichments and isolations were based on a modified 284 mineral salt medium (1.5% agar). 2.Solid media were plated with increasing concent

12、rations of metals. 3.Fresh sediments were resuspended in sterile water (1:1, v/v) and sonicated 3 times for 30 s. After centrifugation of 1 min at 1000 rpm, 50 mL of supernatant were spread on the different solid media. The plates were incubated at 30 Cuntil distinct colonies appeared on the highest

13、 metal concentrations.Strain characterizationAmplify the almost complete 16SrRNA gene (position 29 to 1522 in Escherichia coli). Gene fragments were sequenced by Beckman Coulter Genomics. Genus affiliation of the sequences was then assessed from a BLAST analysis Isolation 1.Total metal levels were l

14、ow in St130 and Ferin, while the most impacted site was Metal-Europ, especially for Cd, Cu, Pb and Zn. Raches had a higher Cu level than MetalEurop.2.It can be seen that the concentration of bioavailable metals was always inferior to the total metal concentration and that a gradient of bioavailabili

15、ty was present from St130 to MetalEurop. In MetalEurop, 9% of the total Zn may be considered as bioavailable; percentages reach 19% for Pb and 8% for Cu.Fig. 2. Total (: above the bars) and bioavailable (black bars) metal concentrations in the top layer (0e1 cm) of Station 130 (St130), Ferin (FER),

16、Raches (RAC)and MetalEurop (MET). HCl-extractable metals (mean SD; left Y-axis) were estimated using 1 M HCl extraction for 24 h. Total metals (mean SD; right Y-axis)were determined by complete digestion in strong acids. Significant differences are indicated by different letters (a, b, c, d).Gene le

17、vels were also plotted against the toxicity index (TI) or the metal pollution index (MPI) and a very clear linear relationship was observed in each case, with the r2 of the linear regression comprised between 0.89 and 0.96These bacteria were essentially -Proteobacteria (58.6%) with Pseudomonas(10 is

18、olates, 34%) and Klebsiella (5 isolates, 17.2%).Five isolates grouped with Firmicutes (Mycobacterium, Streptomyces, Rhodococcus), three with a-Proteobacteria(Sphingomonas and Methylobacterium) and only one with-Proteobacteria (Delftia)Resistances to Cu, Pb and As were the most frequent, and colony f

19、ormation on these metals was observed for most of the 29 isolates (89%). Resistances to Co and Cd were the least frequent and concerned only 5 isolates (17%). For Zn, 11 isolates grew on 1 mM Zn.Discussion 1. The fact that the oprI gene was not found in deep layers of the sediments is probably relat

20、ed to the behavior of Pseudomonas species that are mainly aerobic, and thus mainly located in the top layers of the sediments. In addition, oprI genes are probably not multi-copy genes, contrary to 16S rRNA genes copies in the P. putida genome.2. Our results should, of course, be confirmed in future

21、 studies by other methods such as FISH and/or metagenomics. However, FISH is difficult to apply when sediments contain low levels of cells, cells in biofilms and autofluorescing particles, as in the present case.3.For five of the metals investigated (Cu, Co, Ni, Pb and Zn) gene levels were positively correlated with both total metals and HCl-extractable metals (the potentially “bioavaila