The
Limited work has been conducted to investigate
Soil and water were collected from Newby Ditch in June of 2006 within 24 hours of a rainfall event (1.93 cm or 0.76 inches). Newby Ditch is located within the Mississinewa watershed on the Tipton Till Plain of East Central Indiana (USA). This watershed is characterized as a highly disturbed landscape predominated by row crop agriculture. Environmental samples were placed in sterile bottles and stored in a cooler with ice packs for transport to the lab. The samples were processed within 24 hours for use as soil and water microcosms according to the methods described below.
The stream water used in this experiment had a pH of 7.26. Aliquots of water (90 mL) were placed into 250 mL sterile bottles and autoclaved at
The soil used in this study was a Fox silt loam with a pH of 6.95 [
The initial (zero-time postinoculation) concentration
Total RNA was isolated using the FastRNA Pro Soil-Direct Kit (Qbiogene, Inc., CA) with minor modifications (described below) to improve quality and yield. The kit-supplied Lysing Matrix E tubes were placed at
DNA microarrays were used to evaluate the genetic expression profiles of
The standard protocol for prokaryotic sample and array processing recommended by Affymetrix in their GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA) was used. cDNA was synthesized using a T7 promoter-dT24 oligonucleotide as a primer with the Invitrogen Life Technologies SuperScrip Choice system. Following second-strand cDNA synthesis and incubation with T4 DNA polymerase, the products were purified using the Affymetrix Cleanup Module. Biotinylated cRNA was made using the Affymetrix IVT kit. The cRNA was purified using the Qiagen RNeasy column, quantitated, and then fragmented by incubation at high temperature with magnesium. Biotinylated cRNA was then added to a hybridization solution and hybridized to the GeneChip after adding control oligonucleotides at
The microarray expression data were generated using Affymetrix GCOS software. The Affymetrix Microarray Suite Algorithm was used to analyze the hybridization intensity data from GeneChip expression probe arrays and to calculate a set of metrics to describe probe set performance. The average intensity of each array was normalized by global scaling to a target intensity of 1000. An average expression value for each treatment group was calculated via geometric mean because it is better applied to data with large fluctuations. Only probe sets that received a “present” call of 75% or greater were considered. The expression values were normalized by
Functions of significantly expressed genes were determined using the Affymetrixs NetAffx Analysis Center (
Soil microcosms were inoculated with 10 mL of 8.8
Survival of
Sterile water was inoculated with 10 mL of
Survival of
The genomic expression profiles of
The log expression ratio of the
The log expression ratio of the
An analysis of gene ratios with significant expression levels (
Functional groups differentially expressed between growth in LB and growth in sterile stream water.
Functional group | Total | Higher in LB | Higher in water |
---|---|---|---|
Whole genome | 38 | 26 | 12 |
Antibiotic resistance | 0 | 0 | 0 |
Biosynthesis | 0 | 0 | 0 |
DNA replication/repair, restriction/modification | 2 | 1 | 1 |
Metabolism | 0 | 0 | 0 |
Pathogenesis and virulence | 0 | 0 | 0 |
Phage, transposon, or plasmid | 0 | 0 | 0 |
Ribosomal proteins | 1 | 1 | 0 |
Signaling and motility | 1 | 1 | 0 |
Stress response | 3 | 3 | 0 |
Transcription, RNA processing, and degradation | 4 | 4 | 0 |
Translation and posttranslational modification | 1 | 1 | 0 |
Transport and binding proteins | 7 | 6 | 1 |
Uncategorized | 19 | 9 | 10 |
Functional groups differentially expressed between growth in LB and growth in sterile soil.
Functional group | Total | Higher in LB | Higher in soil |
---|---|---|---|
Whole genome | 397 | 89 | 308 |
Antibiotic resistance | 3 | 0 | 3 |
Biosynthesis | 21 | 1 | 20 |
DNA replication/repair, restriction/modification | 10 | 2 | 8 |
Metabolism | 33 | 15 | 18 |
Pathogenesis and virulence | 7 | 0 | 7 |
Phage, transposon, or plasmid | 8 | 3 | 5 |
Ribosomal proteins | 45 | 0 | 45 |
Signaling and motility | 2 | 2 | 0 |
Stress response | 18 | 0 | 18 |
Transcription, RNA processing, and degradation | 39 | 5 | 34 |
Translation and posttranslational modification | 27 | 2 | 25 |
Transport and binding proteins | 48 | 21 | 27 |
Uncategorized | 136 | 38 | 98 |
A functional group analysis was performed for significantly expressed genes in LB versus sterile soil (Table
Selected genes differentially expressed between growth in LB and sterile soil microcosms.
Function and gene | Description | Logarithmic ratio (S/C) | |
---|---|---|---|
Amino acid biosynthesis | |||
Histidine biosynthesis | 2.05 | ||
Arginine biosynthesis | 3.05 | ||
Arginine biosynthesis | 3.52 | ||
Arginine biosynthesis | 3.07 | ||
Asparagine synthetase B | 3.41 | ||
Thr operon leader peptide | 3.43 | ||
Cysteine biosynthesis | 3.53 | ||
Antibiotic resistance | |||
Multiple antibiotic resistance protein | 4.20 | ||
Multiple antibiotic resistance protein | 4.41 | ||
Multiple antibiotic resistance protein | 5.16 | ||
DNA replication/repair, restriction/modification | |||
Primosomal replication protein | 2.19 | ||
DNA topoisomerase I | 2.33 | ||
DNA binding protein Fis | 2.37 | ||
Primosome assembly protein | 2.60 | ||
Metabolism | |||
Pyruvate dehydrogenase E1 subunit | 2.03 | ||
Protein asmA precursor; electron transport | 2.21 | ||
Dihydrolipoamide dehydrogenase; energy metabolism | 2.24 | ||
N-ethylmaleimide reductase; central intermediary metabolism | 2.71 | ||
Anaerobic glycerol-3-phosphate dehydrogenase subunit C | 2.81 | ||
Cysteine metabolism; amino acid metabolism | 3.25 | ||
Isocitrate dehydrogenase; TCA cycle metabolism | 3.48 | ||
Alcohol dehydrogenase class III; energy metabolism | 4.19 | ||
Phosphoglyceromutase; carbohydrate metabolism | 6.21 | ||
L(+)-tartrate dehydrase; energy metabolism | 0.44 | ||
Formate hydrogenlyase subunit 7; mitochondrial electron transport | 0.44 | ||
Carbon-phosphorus lyase complex subunit; central intermediary metabolism | 0.45 | ||
Acetaldehyde dehydrogenase; amino acid metabolism | 0.46 | ||
Carbon-phosphorus lyase complex subunit; central intermediary metabolism | 0.46 | ||
Hydrogenase 4 Fe-S subunit formate hydrogenlyase, complex iron-sulfur protein | 0.47 | ||
Phosphate acetyltransferase | 0.47 | ||
2,4-dienoyl-CoA reductase (NADPH), NADH and FMN-linked | 0.49 | ||
Pathogenesis and virulence | |||
Ribonuclease R, exoribonuclease R, RNase R | 2.26 | ||
Colicin production | 2.26 | ||
Colanic acid biosynthesis; resistance to acid stress, desiccation, and thermal stress | 2.51 | ||
Hypothetical protein | 2.66 | ||
Phage, transposon, or plasmid | |||
Rac prophage; phage superinfection exclusion protein | 0.43 | ||
Rac prophage; restriction alleviation protein | 0.43 | ||
Rac prophage; conserved protein | 0.44 | ||
Rac prophase; conserved protein | 0.45 | ||
Rac prophage predicted protein | 0.50 | ||
E14 prophage; conserved protein | 4.46 | ||
Translational repressor mprA; plasmid related function | 3.53 | ||
Ribosomal proteins | |||
30S ribosomal protein S21 | 2.25 | ||
30S ribosomal protein S20 | 2.35 | ||
50S ribosomal protein L25 | 2.35 | ||
50S ribosomal protein L32 | 2.43 | ||
50S ribosomal protein L34 | 2.52 | ||
50S ribosomal protein L11 | 2.67 | ||
30S ribosomal protein S18 | 2.87 | ||
50S ribosomal protein L1 | 2.87 | ||
50S ribosomal protein L30 | 3.17 | ||
30S ribosomal protein S10 | 3.24 | ||
50S ribosomal protein L31 | 3.32 | ||
50S ribosomal protein L23 | 3.53 | ||
30S ribosomal protein S8 | 4.34 | ||
50S ribosomal protein L6 | 4.57 | ||
50S ribosomal protein L24 | 4.66 | ||
30S ribosomal protein S14 | 5.68 | ||
Stress response | |||
Cold shock protein E | 2.02 | ||
Cold shock protein, associated with 30S ribosomal subunit | 2.10 | ||
Heat shock protein | 2.10 | ||
SOS cell division inhibitor | 2.17 | ||
Cold shock gene | 2.33 | ||
Heat shock protein; molecular chaperone | 2.48 | ||
Recombinase A; SOS response | 2.59 | ||
Response to organic acid stress and acetate induced acid tolerance; regulatory function | 2.68 | ||
Alkyl hydroperoxide reductase C22 protein; oxidative stress | 2.91 | ||
Cold shock DEAD box protein A | 3.04 | ||
RNA polymerase sigma factor; heat response | 3.19 | ||
Envelope stress induced periplasmic protein | 3.54 | ||
Osmotic adaptation; Osmotically inducible lipoprotein B precursor | 4.77 | ||
Cold shock protein | 5.04 | ||
Envelope stress response | 7.08 | ||
Cold shock protein cspA, major cold shock protein | 8.59 | ||
Transcription, RNA processing, and degradation | |||
DNA directed RNA polymerase beta subunit; transcription | 2.12 | ||
Hypothetical protein, RNA polymerase sigma factor | 2.20 | ||
Polyribonucleotide, nucleotidyltransferase; RNA processing | 2.21 | ||
Ribonuclease P | 2.31 | ||
Ribonuclease E, fused ribonuclease E: endoribonuclease | 2.72 | ||
DNA-directed RNA polymerase alpha subunit; transcription | 2.92 | ||
Transcription elongation factor NusA | 3.16 | ||
Transcription termination factor Rho | 3.34 | ||
Translation and posttranslational modification | |||
Regulatory protein, DNA binding dual transcriptional regulator | 2.44 | ||
Elongation factor Tu, protein chain elongation factor (EF-Tu) | 2.61 | ||
Translation initiation factor IF-1 | 3.09 | ||
Translation initiation factor IF-3 | 3.31 | ||
Elongation factor EF-2 | 3.36 | ||
Translation initiation factor IF-2 | 4.13 | ||
Transport and binding proteins | |||
Phosphonate transport, N-acetyltransferase activity | 0.46 | ||
PTS system, trehalose-specific IIBC component; transport of small molecules | 0.47 | ||
Maltose/maltodextrin transport | 0.48 | ||
Thiamine transport | 0.49 | ||
Iron-enterobactin transporter subunit | 0.49 | ||
Copper efflux system protein | 2.02 | ||
Phosphate transport, sodium dependent phosphate transporter | 2.07 | ||
Leucine/isoleucine/valine transporter subunit | 2.22 | ||
Oligopeptide transporter subunit | 2.32 | ||
Lipoprotein releasing system, transmembrane protein lolE | 2.32 | ||
Preprotein translocase; protein transport | 2.46 | ||
Glutamine ABC transporter, periplasmic-binding protein | 2.50 | ||
Arginine transport | 2.52 | ||
Ferric enterobactin transport system | 3.25 | ||
Ferric enterobactin transport protein | 3.85 |
Selected genes differentially expressed between growth in LB and sterile water microcosms.
Function and gene | Description | Logarithmic ratio (W/C) | |
---|---|---|---|
DNA replication/repair, restriction/modification | |||
DNA restriction-modification system; DNA methylation | 2.38 | ||
Membrane | |||
Outer membrane protein | 0.42 | ||
Outer membrane protein X; integral to outer membrane | 0.43 | ||
Metabolism | |||
Protein yfiD, pyruvate formate lyase subunit | 0.48 | ||
Ribosomal proteins | |||
Protein biosynthesis, structural constituent of ribosome, intracellular ribosome, ribonucleoprotein complex | 0.48 | ||
Regulatory RNA | |||
Regulatory sRNA | 0.23 | ||
Regulatory RNA | 0.39 | ||
Regulatory sRNA | 0.41 | ||
Unknown RNA | 0.46 | ||
Stress response | |||
Cold-shock stress protein | 0.48 | ||
DNA protection during starvation conditions | 0.49 | ||
Transcription, RNA processing, and degradation | |||
Integration host factor alpha subunit; DNA recombination and transcription regulation | 0.49 | ||
Transport and binding proteins | |||
Outer membrane protein F precursor; ion transport, porin activity | 0.46 | ||
Putrescine-binding, periplasmic protein precursor | 2.71 |
The genes responsible for the stress response include those that function in temperature shock, acid tolerance, the SOS response, and osmotic challenge. Eighteen stress response genes were significantly expressed in cells incubated in sterile soil compared to LB (Table
Microarray analysis revealed that cells incubated in sterile soil for 14 days remain very active. In fact, 308 genes were found to be more highly expressed in these cells compared to cells grown in LB. A functional group analysis revealed that the majority of these genes were involved in amino acid biosynthesis, DNA replication and repair, pathogenesis and virulence, the stress response, ribosomal proteins, antibiotic resistance, transcription, and translation. On the other hand, microarray analysis of cells placed in sterile stream water for 14 days revealed that only 12 genes were more highly expressed in this condition. The majority of these genes are uncategorized and of unknown function. There was a marked difference in the expression of ribosomal protein and translation genes. Typically, faster-growing cells synthesize proteins more rapidly and contain more ribosomes [
It is thought that the same mechanism that functions during amino acid starvation also functions during growth rate transitions. In fact, the continued accumulation of RNA in cells under partial amino acid starvation has been shown to be accompanied by a continued synthesis in ribosomal proteins [
The regulatory mechanism that controls the general stress response is the RpoS sigma factor (
The heat shock response is a protective mechanism to cope with heat-induced damage to proteins; however, there is evidence suggesting that these genes are also induced in response to acidic conditions [
Two genes involved in the SOS response were significantly expressed in cells grown in soil compared to LB:
Several genes responsible for the pathogenesis and virulence of
In conclusion, Affymetrix GeneChip
The microarray studies were carried out using the facilities of the Center for Medical Genomics at Indiana University School of Medicine. The Center for Medical Genomics is supported in part by the Indiana Genomics Initiative at Indiana University (INGENE, which is supported in part by the Lilly Endowment, Inc.). In addition, the authors thank Dr. Michael Guebert at Taylor University for assistance in selecting the sample site.