Microbial stress response and survival

1
Isolation and characterization of sulfate
reducing bacteria from NABIR Field Research
Center, TN and heavy metal impacted lake.
Sergey Stolyar, Beto Zuniga, Martin Koenneke,
Nick Pinel, Zhili Heb, Qiang He b, Jizhong
Zhoub, Terry Hazena, Sharon Borglina, David Stahl
University of Washington, Seattle WA 98195 a
Lawrence Berkeley National Lab CA b Oak Ridge
National Lab TN
UW
Abstract Isolation and characterization of SRBs
from FRC. MPN enrichments were set up
using sediments (FWB203-03d 04) from FRC area 2
(17.5' to 22.5' depth ) in PIPES buffered B2
medium supplemented with one of the following
substrates 1) lactate, 2) lactate plus ethanol,
3) acetate, 4) propionate 5) pyruvate or 6)
hydrogen with carbon dioxide. Growth was
observed on all substrates in the 10-1 to 10-4
dilutions. All of these MPN enrichments showed
sulfate reducing activity (Fig. 2, 3 ). Thirty
isolates from the lactate containing medium were
recovered. All of these isolates demonstrated
slow growth at 25C. Analyses of 16S rRNA gene
sequences amplified from 12 isolates were
identical and affiliated with gram Firmicutes
from genera Clostridium and Dendrosporobacter.
Another culture started from an enrichment using
a defined multipurpose medium (Widdel and Bak,
1992) supplemented with H2, CO2 and 1mM acetate
demonstrated growth and sulfate reducing activity
after several consecutive transfers in defined
liquid medium. Light microscopy revealed that the
majority of cells in this culture were long,
thin, bended gram-negative rods, often appearing
as chains (Fig. 3). Isolation and
characterization of Desulfovibrio-like organisms
from heavy metal impacted lake.
Sediment from a metal contaminated Lake (Lake
DePue, Illinois) was enriched for
sulfate-reducing bacteria using a defined
multipurpose medium (Widdel and Bak, 1992).
Three carbon sources plus sulfate were evaluated
lactate, acetate/H2/CO2, and propionate.
Growth and sulfide production was observed with
lactate and acetate/H2/CO2 media. Nine colony
isolates were recovered from the lactate
enrichment using agar roll tubes, transferred to
liquid medium and isolated as pure cultures (
isolates DP1-9). Their 16S rRNA and dsrAB genes
were amplified and sequenced. All sequences were
identical to each other and virtually identical
to the corresponding genes from D. vulgaris
Hildenborough (DvH). One mismatch was observed
in the16S rRNA gene and one in dsrAB. Pulse field
electrophoretic analysis of I-CeuI digests
revealed that isolates had five rRNA clusters,
the same as DvH. However, the length of one
chromosomal segment in the DP isolates was
considerably shorter than the corresponding
fragment from DvH, suggesting the presence of a
large deletion in the genomes of the isolates.
Comparative whole-genome microarray hybridization
analysis of DNA isolated from isolate DP4 and
was conducted using D. vulgaris Hildenborough
oligo microarray. We found more than 300 genes
of DP4 isolate that did not hybridized to the
microarray were mostly distributed in seven
regions on the chromosome of DvH . Most of them
were annotated as genes for conserved/
hypothetical or phage related proteins (Table 1).
The growth rates at different temperatures on B3
medium supplemented with lactate and sulfate were
comparable for all DP isolates tested. However,
biomass yield was always lower than that of DvH
(Fig. 7). In addition, resistance of isolates
from Lake DePue (DP1-9) and DvH to ZnCl2 growing
in LS4D medium were assessed using Omnilog System
and revealing no difference among all isolates
and DvH (Fig. 8). Widdel, F. and Bak, F., 1992.
Gram-Negative Mesophilic Sulfate-Reducing
Bacteria. In Balows, A., Trüper, H.G., Dworkin,
M., Harder, W. and Schleifer, K.-H.(Eds.), The
Prokaryotes, 2nd ed. New York, Springer-Verlag.
pp. 3352-3378.
Characterization of isolates from heavy metal
impacted lake
Figure 6. D. vulgaris Hildenborough chromosome
map and gene clusters deleted in the DP4 isolate
genome
Figure 5. Pulse field gel electrophoresis analysis
Table 1. Summary of gene content in the potential
deletion regions in Desulfovibrio DP4 strain
compared to D. vulgaris using the whole-genome
oligonucleotide array of D. vulgaris Hildenborough
1- Size markers (Saccharomyces cerevisiae) 2,
10- D. vulgaris Hildenborough (DvH) (undigested
genomic DNA) 3, 11- L. DePue isolates DP4 and
DP8 undigested genomic DNA 4- DvH DNA digested
with I-CeuI 5,6- DP4 and DP8 DNA digested with
I-CeuI 7- DvH gDNA digested with NotI 8,9- DP4
and DP8 gDNA digested with NotI Arrows indicate
extrachromosomal (plasmid) DNA red diamonds
indicate I CeuI fragments of DvH
Figure 1. Phylogenetic neighbor joining analysis
of 16S rDNA sequences. Scale bar represents 0.1
nucleotide change per position.
Conclusions
Characterization of isolates from FRC sediment

• Growth of MPN enrichments inoculated with
  sediments from FWB203-03d 04 from FRC area 2 was
  observed on 1) lactate, 2) lactate plus ethanol,
  3) acetate, 4) propionate 5) pyruvate or 6)
  hydrogen with carbon dioxide in the 10-1 to 10-4
  dilutions (Fig. 2).
• 30 isolates from the lactate containing medium
  only were recovered as colonies in agar medium.
  Analyses of 16S rRNA gene sequences amplified
  from 12 of these isolates showed that they were
  identical and affiliated with Fimicutes (Fig. 1).
• One culture from an enrichment supplemented with
  H2, CO2 and 1mM acetate demonstrated growth and
  sulfate reducing activity in several consecutive
  transfers in Widdel Bak defined liquid medium.
  Light microscopy revealed that the majority of
  cells in this culture are long, thin, bended,
  gram-negative rods (Fig. 3).
• Nine colony isolates were recovered from the Lake
  dePue lactate enrichment using agar roll tubes.
  Their 16S rRNA and dsrAB genes were identical to
  each other and virtually identical to the
  corresponding genes from D. vulgaris
  Hildenborough (Fig. 1).
• About 300 genes of isolate DP4 did not hybridized
  to the D. vulgaris microarray. Thes were mostly
  distributed in seven regions on the chromosome of
  DvH . Most of them were annotated as genes for
  conserved/ hypothetical or phage related (Table
  1 Fig. 6).
• Growth at different temperatures in a B3 medium
  supplemented with lactate and sulfate
  demonstrated that growth rates of DP isolates
  were comparable with those of DvH. However,
  isolates biomass yield was always lower than that
  of DvH (Fig. 7).
• Resistance of isolates from Lake DePue (DP1-9)
  and DvH to ZnCl2 growing in LS4D medium were
  similar for all isolates tested and DvH (Fig. 8).

Figure 3. Phase contrast micrographs of
enrichment FRC2SHA_1 and FRC2LacA1
Figure 4. Identification of metabolites excreted
by FRC2LacA1_24 using anion chromatography on
Dionex and HP 5972 GC/MS.

Figure 8. Growth of DvH and DP1 isolates in the
presence of different concentrations of ZnCl2 in
LS4D medium using an Omnilog System.
FRC2LacA1_24
FRC2LacA1_30
FRC2SHA_1
FRC2SHA_1
Acknowledgments
FRC2LacA1_24 was grown in B3 medium supplemented
with 20mM lactate and 10mM sodium sulfate at
25oC without shaking. Peaks were identified was
done using MS by Dr. M. Sadilek (UW, Department
of Chemistry ).
This work was part of the Virtual Institute for
Microbial Stress and Survival supported by the US
Department of Energy, office of Science, Office
of Biological and Environmental Research,
Genomics Program GTL through contract
DE-AC03-76SF00098.
FRC2SHA enrichment was performed in 150ml bottles
by adding 2ml of sediment into 50 ml defined
carbonate buffered media using H2/CO2 (8020
v/v) in the headspace (Widdel and Bak, 1996).
The enrichment were incubated at 30oC without
shaking.
Figure 2. Enrichments and recovering of
FRCLacA1-30 isolates from FRC area 2 sediment.
Enrichment was performed in 25 ml tubes in
defined PIPES buffered B2 medium (Stolyaret al.,
unpublished). The enrichments were incubated at
25 and 30oC without shaking.