| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Matforsk, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway
2 The Norwegian University of Life Sciences (UMB), PO Box 5003, N-1432 Ås, Norway
Correspondence
Askild Holck
askild.holck{at}matforsk.no
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
The array data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE7273.
A complete table showing genes up- and down-regulated in S. aureus after acid shock is available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). It is also a major food poisoning bacterium posing a great risk to consumer health, mainly through its production of heat-stable enterotoxins. S. aureus is considered to be a poor competitor in complex microbial populations. Therefore, the greatest risk of staphylococcal food poisoning is associated with food contaminated with S. aureus after the normal microflora has been destroyed. Although S. aureus is found in many environments, the source of contamination is often human, since a large part of the healthy human population carry the bacteria as part of the normal microflora associated with the nose, throat and skin.
This work constitutes a part of our effort to study different stress responses of S. aureus in food. Acidification is one of the major ways to reduce growth of bacteria in food processing, and S. aureus is quite acid tolerant. In general, bacteria possess a multitude of defence mechanisms to cope with a sudden drop in pH (for a review for Gram-positive bacteria, see Cotter & Hill, 2003). The most direct approach is the use of proton pumps, which literally pump protons out of the cell to keep the internal pH at an acceptable level. Another approach is to increase the concentration of alkaline compounds within the cell to counteract the acidification of the cytoplasm. Repair mechanisms are needed due to an increased rate of damage to macromolecules. Biofilm formation can also affect the acid resistance of the cells. Energy metabolism and metabolic pathways are altered by the shift of energy usage from mainly growth to defence mechanisms.
In contrast to the situation for Escherichia coli, much less is known about the general stress responses in S. aureus. Although some studies have shown the involvement of specific genes or genetic systems, few overall studies have elucidated the general mechanisms employed by S. aureus to cope with acidic environments. Most published work on how S. aureus, or similar bacteria such as Streptococcus, responds to low pH have chosen a steady-state approach, studying growth at low pH in parallel with growth under neutral-pH conditions (Len et al., 2004; Weinrick et al., 2004; Wilkins et al., 2002). In this work, we have chosen to focus on the genetic expression of a food poisoning S. aureus strain along a time curve through the first 20 min after a sudden acid stress to pH 4.5 of exponentially growing cultures. By using a microarray approach in combination with a novel improved statistical analysis, we aspire to give a general overview of how this important pathogen responds to cope with this sudden change of environmental conditions.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
This procedure was repeated on three different days for a total of three sample sets (in parallel). Separate array experiments were performed on all three parallel sample sets.
RNA extraction.
Total RNA was extracted by using the RNeasy Protect Bacteria Mini Prep kit (Qiagen). Briefly, 1.5 ml bacterial culture was added directly to 3 ml RNAprotect Bacteria Reagent, vortexed for 5 s, incubated at room temperature for 5 min and then centrifuged at 10 000 g for 10 min at room temperature. The supernatant was discarded and the pellet resuspended in 40 µl Tris/EDTA buffer followed by addition of 50 µl lysozyme stock solution (40 mg ml–1) and 10 µl lysostaphin stock solution (1 mg ml–1). The samples were incubated at 37 °C with shaking for 10 min. The manufacturer's protocol, including use of kit buffers, was followed from this point. RNase-free water was added to the membrane of the RNeasy Mini Spin column (Qiagen), and after 1 min incubation at room temperature the RNA was eluted by centrifugation of the column at 10 000 g for 1 min. The concentration and purity of the total RNA were analysed with an Ultrospec 3000 spectrophotometer (Pharmacia Biotech) and the RNA 600 Nano LabChip system (Agilent Technologies).
Labelling of total RNA and microarray hybridization.
S. aureus genome microarrays (version 1) were kindly provided by The Institute for Genomic Research (TIGR). The slides contained PCR products of 2576 ORFs from S. aureus COL. In addition the arrays contained 117 unique ORFs from strains Mu50 (60), MW2 (51) and N315 (6). Total RNA was reverse-transcribed by using random hexamers in the presence of aminoallyl-dUTP, followed by labelling with Cy3 and Cy5 monoreactive dyes (TIGR protocol, standard operating procedure no. M007; http://pfgrc.tigr.org/protocols/M007.pdf). Briefly, 2 µg total RNA and 6 µg random hexamers (Invitrogen) in a reaction volume of 18.5 µl were denatured at 70 °C for 10 min, snap-cooled on ice for 30 s and centrifuged briefly to bring down condensation. Then 6 µl First Strand buffer (Invitrogen), 3 µl 0.1 M DTT, 1.2 µl 12.5 mM deoxynucleoside triphosphate-aminoallyl-dUTP labelling mix (Invitrogen) and 400 U Superscript II reverse transcriptase (Invitrogen) were added. The labelling reaction mixture was incubated at 42 °C overnight. The first-strand synthesis reaction was stopped by adding 10 µl 0.5 M EDTA and 10 µl 1 M NaOH. The reaction mixture was incubated at 65 °C for 15 min, and then 25 µl 1 M Tris/HCl (pH 7.0) was added. The removal of unincorporated aminoallyl-dUTP and free amines was performed according to the TIGR protocol with the Qiagen QIAquick PCR purification kit. After being dried in a Speed Vac (Savant), the samples were stored at –80 °C. Coupling of aminoallyl-labelled cDNA to CyDye Ester (Amersham Biosciences) was done according to the TIGR protocol. The eluted Cy3- and Cy5-labelled samples were mixed and dried in a Speed Vac. The hybridization of the labelled cDNA probes was performed according to a protocol from TIGR (standard operating procedure no. M008; http://pfgrc.tigr.org/protocols/M008.pdf). Briefly, slides were incubated in a preheated prehybridization buffer [5x SSC (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1 % SDS, 1 % BSA], at 42 °C for at least 1 h. After prehybridization, the slides were washed in distilled water (five times 2 min in 50 ml tubes) and then in 50 ml isopropanol. The slides were dried by centrifugation at 90 g for 12 min at room temperature. The dried Cy3- and Cy5-labelled cDNA was resuspended in 60 µl hybridization buffer (50 % formamide, 5x SSC, 0.1 % SDS, 300 µg salmon sperm DNA), denatured at 95 °C for 5 min, vortexed at low speed and heated again at 95 °C for 5 min. The sample was centrifuged briefly and applied to the prehybridized microarray under a coverslip (LifterSlip, Erie Scientific). The slides were placed in a waterproof hybridization chamber for hybridization in a 42 °C water bath overnight. After hybridization, the slides were washed in 2x SSC buffer with 0.1 % SDS (55 °C, 2 min), then washed in 0.1x SSC with 0.1 % SDS buffer (room temperature, 2 min), followed by two washes in 0.1x SSC buffer (room temperature, 2 min). The slides were dried by centrifugation at 90 g at room temperature for 12 min.
Reverse transcription and quantitative reverse transcriptase PCR (QRT-PCR).
Total RNA was reverse-transcribed by using random primers. The reaction mixture (20 µl), containing 300 ng total RNA, 100 ng random primers (Invitrogen) and 1 µl 10 mM dNTP mix in a reaction volume of 13 µl, was denatured at 65 °C for 5 min, snap-cooled on ice and centrifuged briefly. Then 4 µl 5x First Strand buffer (Invitrogen) and 2 µl 0.1 M DTT were added and the reaction was incubated at 42 °C for 2 min before 1 µl (200 U µl–1) SuperScript II reverse transcriptase (Invitrogen) was added. The reaction was incubated at 25 °C for 10 min and 42 °C for 50 min. The reaction was centrifuged briefly and incubated at 70 °C for 15 min before being centrifuged briefly again. cDNA was synthesized from two biological parallel samples grown on different days. Three reverse-transcriptase reactions were made for each biological replicate sample of RNA in addition to one negative control (without enzyme). After the reverse-transcriptase reaction, the cDNA was added in 30 µl H2O, and 1.5 µl was used as template in the QRT-PCR analyses. Primers and probes (Table 1) were designed using Primer Express (version 1.0) ABI PRISM (Applied Biosystems) software. The probes contained 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (6-FAM) linked to the 5' end, and tetramethyl-6-carboxyrhodamine (TAMRA) at the 3' end. Amplification efficiency was analysed by performing PCR on dilution series of DNA. The amplification reaction mixture (25 µl) contained: 1.5 µl template cDNA (1000-fold diluted for the 16S PCR); 1x TaqMan buffer A; 5 mM MgCl2; 200 µM each of dATP, dCTP and dGTP, and 400 µM dUTP; 80 µM AmpErase uracil N-glycosylase; 0.2 µM forward and reverse primer; 0.1 µM MGB probe; and 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems). Before amplification, the reaction mixture was heated to 50 °C for 2 min and then denatured at 95 °C for 10 min. The amplification profile was as follows: 40 cycles of 95 °C for 15 s and then 60 °C for 1 min. Reactions were performed in the ABI PRISM 7900HT Sequence Detection system and data analysed using the SDS 2.2 software (Applied Biosystems). 
CT was calculated as the CT value from the unstressed control cells minus the CT value from the stressed cells and corrected for differences in signals from the 16S or gyrB controls.
|
The data were evaluated statistically according to a recently developed approach (Langsrud, 2005) for analysing experiments designed with multiple responses and the calculations were made using the Matlab program at www.matforsk.no/ola/program.htm.
To detect significant differences, all four time points need to be modelled simultaneously. Our analysis was based on the second-order model:
 |
The constant term, A, represents the mean difference between the control and the four time points. An ordinary significance test of A would, however, give misleading results. The reason is that we have a common (not replicated) control at the four time points and therefore the SD of A is underestimated. It can be shown that a conservative estimate can be obtained by scaling the SD by a factor of 
. We applied this property to perform significance testing of A in a conservative way. The significance tests of C (linear trend) and D (deviation from linearity) were conducted as usual.
However, with several multiple responses, ordinary significance testing leads to a large number of falsely significant results (5 % of the total number of genes). Therefore, to draw reliable significance conclusions, the P value of each gene was adjusted using a newly developed rotation-testing procedure; see Appendix in Moen et al. (2005). This method combines general linear modelling with P value adjustments according to false-discovery rates (FDRs). When using adjusted P values according to the FDR, one accepts that 5 % of the genes reported as significantly differentially expressed at the 5 % level are false results (31 of 622 genes). Compared to other FDR variants, an advantage of the rotation-testing method is that any kind of dependence between the genes is allowed.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
. Control samples for microarray analyses were withdrawn immediately prior to the acid shock that lowered pH of the culture to 4.5, and this was defined as time zero. The other samples for microarray analysis were withdrawn 2, 5, 10 and 20 min after the acid shock. In the microarray analyses, a total of 622 genes showed a statistically significant (at 5 % FDR) change in expression level over the four time intervals measured. This result was due to significant mean differences and/or significant linear trends (log scale). No log ratios of gene expression showed significant deviation from linearity in the statistical model. Of the significant gene responses, 311 genes were upregulated and 311 genes down-regulated (Table 3). In general, most of the changes occurred within the first 10 min, and for most of the responses the expression levelled out between 10 and 20 min after the acid shock (Fig. 2). The acid shock reduced the growth rate of the culture and the genetic expression profiles showed this clearly through the down-regulation of the biosynthesis of many macromolecules (Table 3, and see below). Of the genes showing a statistically significant change in expression, 59.6 % showed a maximum change of expression larger than twofold, 23.3 % were larger than threefold, 10.5 % were larger than fourfold and 5.6 % were larger than fivefold. Relatively few genes showed an aberrant pattern, a very fast upregulation or a very fast down-regulation, and none of these is commonly reported to be involved in acid-stress responses. We cannot address all responses in this paper, but an outline of the major responses observed is given. It is important to bear in mind that one cannot expect to see full correlation between gene expression levels and the corresponding activity of the gene product. Likewise, genes showing the highest fold-change might not coincide with the most important genetic response. Generally, one might expect an accumulation of many of the proteins for which gene expression was unchanged in the cell after acid shock, since growth was significantly reduced. No experiments to determine the extent to which this occurred were carried out, however. The literature on stress mechanisms in S. aureus is relatively sparse. Our results (Table 3) closely resembled responses described in the literature on acid stress in S. aureus or other bacteria. We also observed a number of additional responses.
|
|
|
). The two reference genes gave similar results. The QRT-PCR analysis agreed well with the microarray data and provided independent verification of changes in the transcript levels of the genes that we discuss in this paper.
|
, section F3). This result was similar to other findings where S. aureus was grown at pH 5.5 or the bacterium was exposed to 1 mM peracetic acid (Weinrick et al., 2004; Chang et al., 2006, respectively). Urease is a multimeric, nickel-containing enzyme that consists of six UreA and six UreB subunits. Urease activity is known to be the major acid-resistance mechanism in Helicobacter pylori, which has adapted itself to live in the strongly acidic environment of the human stomach. In H. pylori, urease is expressed at very high levels, and the main role of the enzyme is thought to be neutralization of acidic microenvironments by production of NH3 and CO2. The NH3 produced can act as a buffer leaving the bacterial cytoplasm and entering the bacterial periplasm, by the formation of NH4+ (Valenzuela et al., 2003). Urease is expressed by a wide variety of bacteria, although not at the high levels seen in H. pylori. In other bacteria, the urease activity is regulated in response to environmental changes such as changes in pH, urea and nitrogen availability, and growth phase (Burne & Chen, 2000; Chen et al., 1998). In being a nickel metalloenzyme, urease requires active uptake of nickel ions from the environment to maintain its activity. The nixA gene, encoding a nickel transporter necessary for full urease activity (Bauerfeind et al., 1996; Davis & Mobley, 2005; Mobley et al., 1995; van Vliet et al., 2001), was found to be upregulated in our study at a similar level of expression as the urease genes (maximum 5.8-fold after 10 min). Enhanced urease activity therefore seemed to be an important acid-shock mechanism for S. aureus 50583 to counteract the acidic environment.
The arginine deiminase (ADI) pathway generates alkaline compounds to help raise the cellular pH as a response to acid shock in many bacteria (Cotter & Hill, 2003). Of the three main enzymes in the ADI pathway, only arcC2 encoding carbamate kinase showed any significant change in our data, and this gene was down-regulated (minimum 0.61), suggesting that the ADI pathway is not a major acid-shock response in the S. aureus 50583 strain under the conditions given. A similar down-regulation of arcC2 has been observed in peracetic acid-treated cells (Chang et al., 2006).
Proton efflux.
The gene nuoF encoding an NADH dehydrogenase was found to be upregulated (maximum ratio of 4.76 after 10 min). This dehydrogenase moves 2H+ across the membrane and out of the cell, converting one NADH to NAD+, and thereby helps to increase the internal pH of the cell. Somewhat surprisingly, we found that the F0F1 ATPase was down-regulated at pH 4.5. All genes encoding subunits for both F1 (
, β, 
, 
and 
) and F0 (a, b and c) were down-regulated to minimum ratios of the order of 0.37–0.53 (Table 3, section D2). In the literature, F0F1 ATPases are often mentioned as one of the primary mechanisms for extruding protons in many Gram-positive bacteria (Foster, 2004). Len et al. (2004) have shown increased levels of F0F1 ATPase at pH 5.0 in Streptococcus mutans. On the other hand, microarray analysis of S. aureus does not show any significant change in expression of the F0F1 ATPase genes when cells are grown at pH 5.5 or subjected to 1 mM peracetic acid (Weinrick et al., 2004; Chang et al., 2006). In addition, recent work by Maurer et al. (2005) has shown that in E. coli, the ATPase genes are induced at high pH rather than at low pH. The genetic expression was clearly reduced to about 50 %, but the ATPase could still play an important role by pumping H+ out of the cells, since regulation of ATPase activity apparently occurs at the assembly stage (Arikado et al., 1999). The gene SACOL0687, encoding a Na+/H+ antiporter, was upregulated.
Reducing power.
As the acid shock leads to increased consumption of reducing power in the form of NADPH and NADH, an urgent need to generate more reducing power arises in bacterial cells. For NADPH, this need can be met by increased flow through the pentose phosphate pathway (PPP). Several genes encoding enzymes in the PPP and in the overlap between the PPP and glycolysis were upregulated after the acid shock (zwf, gnd, tkt, pgi, fdaB). None of these genes has been reported to show changes in expression when cells are grown at pH 5.5 (Weinrick et al., 2004). The gene zwf, encoding the glucose-6-phosphate dehydrogenase, is the first irreversible enzyme leading into the PPP. Together with gnd (6-phosphogluconate dehydrogenase) it controls the carbon flow into the PPP. Both these genes were found to be upregulated in our study (zwf 3.1-fold and gnd 2.1-fold after 10 min). Upregulation of zwf as a response to oxidative stress and recovery from heat stress has been demonstrated for Gram-negative bacteria (Cohen et al., 1993; Greenberg et al., 1990; Kobayashi et al., 2005; Ma et al., 1998).
Several genes encoding enzymes in the tricarboxylic acid (TCA) cycle were also upregulated after the acid shock (sucA, sucB, icd, gltA, mqo1). The sucA and sucB genes in the TCA cycle encode 2-oxoglutarate dehydrogenase. It has been proposed that this complex may provide succinyl-CoA for phosphorylation of GDP and ADP under conditions of restricted NAD+ availability, which could be essential upon accumulation of NADH and exhaustion of the pyridine nucleotide pool (Bunik, 2003).
Several genes involved in conversion of acetate to ethanol were upregulated after the acid shock. Among these were four zinc-containing alcohol dehydrogenases, two aldehyde dehydrogenases (AldA1, AldA2), and -acetolactate decarboxylase (BudA1) and acetolactate synthase (BudB). Increased activity of these dehydrogenases would accelerate the regeneration of NAD+, and one might also speculate that this could raise the intracellular pH by the removal of acetate. Len et al. (2004) have reported a 4.6-fold higher level of ethanol produced by Strep. mutans grown at pH 5.0 compared with pH 7.0.
Among other energy-metabolism genes showing a significant increase in expression was an acetyl-CoA synthetase (SACOL2624), which could generate acetyl-CoA from acetate for use in the TCA cycle. The two genes bud A1 and budB (maximum upregulation 5.7- and 4.8-fold, respectively) are also part of the C5-branched dibasic acid metabolism pathway and encode enzymes that produce acetoin. It is known that conversion of pyruvate to acetoin contributes to maintenance of pH homeostasis in Lactobacillus plantarum (Tsau et al., 1992).
Regulators.
Several regulatory genes play a role in the exponential-phase acid-tolerance response. Among these, rsbU was found to be down-regulated, while phoP and SACOL1541 (transcriptional regulator of the iron regulator Fur family) were found to be upregulated (Table 3, section G). Also, many other transcriptional regulators showed a significant change of expression in our data (Table 3, section G). The rsbU gene encodes the 
B regulator RsbU, which is thought to be the major activator of the B response to acid stress in S. aureus (Palma & Cheung, 2001). The down-regulation of this gene is in agreement with the lack of any significant increase for B. B partially controls another well-studied signal-transduction regulatory component, sarA (Deora et al., 1997; Manna et al., 1998; Miyazaki et al., 1999), the expression of which remained unchanged after the acid shock. In Salmonella enterica, S, PhoP and Fur are known to be induced by acid shock to pH 4.5, and rpoS, phoP and fur mutants are known to be acid sensitive (Foster, 2004). Besides phoP, two genes involved in different two-component systems were upregulated (kdpD and vraS), while two were down-regulated (saeS and saeR). The latter two genes are known to be important for virulence (Novick & Jiang, 2003), while KdpD is involved in sensing K+ levels (Epstein, 2003; Wood, 1999) and VraS is involved in the cell wall stimulon response (Kuroda et al., 2003).
Indirect responses
Oxidative stress response.
The ability to defend against oxidative stress is of vital importance to S. aureus when attacked by host phagocytes at the site of bacterial infection. Oxidative-defence mechanisms are also generally important for bacterial survival in the environment, and the enzymes involved may provide the bacteria with a defence against oxidative threats such as the disinfectants employed in the food industry and in hospitals. Our results showed upregulation of the superoxide dismutase (SOD) gene sodA and the catalase gene katA, thus connecting the acid response to the oxidative-stress response. The SodA protein can reduce the rate of neutrophil-mediated killing and destroys highly reactive 
(Clements & Foster, 1999). Adaptation of S. aureus to acid leads to induction of sodA, and sodA mutants are known to be more sensitive to acid (Clements et al., 1999). KatA is the only major catalase in S. aureus and accumulates extracellularly, converting H2O2 to water. When cells are grown at pH 5.5, no increase in katA and sodA expression has been reported (Weinrick et al., 2004). Somewhat surprisingly, this is also the case when cells are subjected to 1 mM peracetic acid (Chang et al., 2006).
Our results also showed an upregulation of the genes ahpC and ahpF that encode the alkyl hydroperoxide reductase subunits C and F, respectively. Alkyl hydroperoxide reductase (AhpC) protects cells against OONO–, which is also generated within neutrophils and macrophages. The gene ahpC has been shown to be induced by osmotic stress but not by oxidative stress in S. aureus (Armstrong-Buisseret et al., 1995). Regulation of katA and ahpC via perR has been described previously (Horsburgh et al., 2001).
Three thioredoxin genes were shown to be upregulated in our data (trxA, trxB and SACOL1794). Thiol-specific redox systems are vital for the protection of cells against toxic oxygen species. They also play a major role in maintaining the intracellular thiol–disulfide balance and in providing reducing power to the key reductive enzymes, such as ribonucleotide reductases. In many prokaryotes, thioredoxin and glutaredoxin execute these tasks. S. aureus lacks glutathione and must therefore employ thioredoxin and alternative thioredoxin-based systems, such as alkyl hydroperoxide reductases, to carry out these cellular processes (Hofmann et al., 2002). Thioredoxins are small ubiquitous proteins participating in numerous dithiol–disulfide interchange reactions. One of the established functions of thioredoxins is to reduce disulfide bonds in target proteins. The reducing power for this is provided by thioredoxin reductase, which in turn is reduced by NADPH. Thioredoxin is also essential for growth (Uziel et al., 2004). Linkage between acid and oxidative responses has also been suggested by others, for both S. aureus and E. coli (Clements & Foster, 1999; Clements et al., 1999; Maurer et al., 2005).
Metal transporters.
In general, the expression of many genes that encode transporters was significantly changed after the acid shock, and that of ABC transporters in particular. The gene SACOL2572, encoding a copper-translocating P-type ATPase, was upregulated. The copper-translocating P-type ATPase can pump excess Cu2+ ions out of the cell at the expense of ATP. Low pH can increase metal ion toxicity in bacteria, and an excess of metal ions causes oxidative damage. The sensing and regulation of metal ion levels are therefore essential for the cell in a stressed situation. The merR regulators are known to be involved in the regulation of heavy metal ion concentrations in many bacteria (for a review see Brown et al., 2003), and two of these merR regulator genes (SACOL2193 and SACOL2517) were upregulated in the acid-shocked cells.
Genes encoding transporters of metal ions, such as nickel, iron and manganese, as well as genes encoding ABC transporter systems for the transport of molybdenum and zinc into cells, were upregulated after the acid shock (Table 3, sections B2 and B5). One might speculate that this is due to an increased need for these essential metal ions in enzyme systems (Kisker et al., 1997). As stated above, several zinc-containing alcohol dehydrogenase genes and the nixA gene encoding a nickel transporter were found to be upregulated. Likewise, eight genes involved in molybdopterin cofactor synthesis were upregulated. In the Gram-positive bacterium Streptomyces coelicolor, others have linked molybdopterin biosynthesis to disulphide stress caused by the thioredoxin activity (see above) which follows from oxidative stress (Paget et al., 2001).
Other transporters.
Several genes encoding transporters of osmoprotectants were found to be upregulated. Down-regulated transporter genes included those encoding transporters for formate and lactate, spermidine/putrescine, sugars and amino acids. The reduction in expression of transporters for spermidine and putrescine is interesting, since these are alkaline compounds that might contribute to keeping a higher pH inside the cell.
DNA-repair systems.
It is well known that low pH causes damage to DNA. One would therefore expect expression changes in genes involved in DNA replication and repair.
DNA-damage-inducible protein P (dinP) showed an early increase in expression. Also, several genes involved in DNA replication and repair, such as polA, encoding DNA polymerase I, and rexA and rexB, encoding the exonucleases RexA and RexB, respectively, were found to be upregulated. In Gram-positive bacteria, the exonuclease/helicase RexAB is a major component of the homologous recombination process and is essential for double-strand DNA break repair (Chedin & Kowalczykowski, 2002; Quiberoni et al., 2001). On the other hand, other genes involved in DNA repair and replication were found to be down-regulated, e.g. DNA-repair protein RecN and exonuclease SbcD. The gene priA, encoding a helicase (Lasken & Kornberg, 1988), was found to be upregulated. Others have shown that inactivation of this gene induces the SOS response in E. coli (Nurse et al., 1991).
Changes related to metabolism and growth.
The lower growth rate of the bacterium after the acid shock was reflected in the down-regulation of a high number of genes involved in protein synthesis, as well as in fatty acid and phospholipid metabolism. A total of 42 ribosomal protein genes showed significant down-regulation after the pH drop (Table 3, section E2). Only two were upregulated (rpmG and rpsA). In addition, frr, encoding ribosome recycling factor, was down-regulated. The level of down-regulation was in the range 0.25–0.63 with an average of 0.46, with a general tendency for a gradual decrease from time zero to 20 min. We also found several other genes involved in protein synthesis to be down-regulated, including translation factors, tRNA and rRNA base modification genes, and genes linked to tRNA aminoacylation. Among these, five tRNA synthetase genes (tyrS, pheS, argS, gltX and lysS) were shown to be down-regulated, while cysteinyl- and leucyl-tRNA synthetases (CysS and LeuS) were upregulated.
Likewise, the lower growth rate resulted in down-regulation of seven and 14 genes, respectively, of the pyrimidine and purine ribonucleotide biosynthesis pathways (Table 3, sections A1–A2). The same was also the case for seven genes involved in nucleotide and nucleoside interconversions (Table 3, section A3). While most of the PPP was upregulated to generate reducing power, the gene prsA (ribose-phosphate pyrophosphokinase), catalysing the first step from D-ribose-5-phosphate from the PPP into the pyrimidine and purine synthesis pathways, was found to be down-regulated. Also the regulator gene pyrR, encoding pyrimidine operon regulatory protein, showed a major down-regulation in expression level after the acid shock.
In contrast to the major down-regulation of genes involved in protein and nucleotide synthesis, we found that a number of genes involved in the degradation of proteins, peptides and glycopeptides were upregulated at relatively high ratios (Table 3, section E5). Four genes involved in protein modification and repair were also upregulated. The upregulation of these types of genes could be a result of both an increased need for the degradation of proteins subjected to acid denaturation and inactivation, and a need for amino acids for the increased synthesis of proteins involved in the acid response. Three peptidases were also found to be down-regulated (M16 family and U32 family).
Our results indicated that both clpB and clpC, encoding subunits of an ATP-dependent Clp protease, are part of an acid-shock response in S. aureus 50583, as we saw a major upregulation with maximum expression 10 min after the acid shock (8.2-fold for clpB and 3.6-fold for clpC), followed by a slight decrease in expression from 10 to 20 min. This decrease is consistent with the increase in expression of the ctsR gene, encoding a negative heat-shock regulator known to control expression of clpB and clpC (Frees et al., 2004). Those authors have shown that ClpC is required for the repressor activity of CtsR, and suggest that ClpC is a stress sensor for S. aureus during environmental stress and infection (Frees et al., 2004). The gene SACOL2563, encoding a putative ATP-dependent Clp protease, was also found to be upregulated after the acid shock. Others have recently shown that S. aureus ClpC is required for resistance to stresses such as oxidative and heat stress, aconitase activity, growth recovery and death (Chatterjee et al., 2005; Frees et al., 2004), as well as biofilm formation and thermotolerance, in which ClpB also plays an important role (Frees et al., 2004). In Listeria monocytogenes, clpB has been linked to virulence and shown not to be involved in stress tolerance (Chastanet et al., 2004). Upregulation of clpB in S. aureus exposed to acid has also been reported by Weinrick et al. (2004) at steady state after 6 h growth. The gene SACOL1777, encoding HtrA serine protease, was also found to be upregulated. In E. coli, the HtrA protease is known to be heat-shock induced, and its main role is to degrade misfolded proteins in the periplasm (Pallen & Wren, 1997).
Genes encoding enzymes in the major energy metabolic pathway, glycolysis, generally showed only minor changes in expression after the acid shock. The exception was the gene fdaB, encoding fructose-diphosphate aldolase, which showed an increase in expression of up to 7.1-fold after 20 min. The genes pgi (glucose-6-phosphate isomerase) and eno (enolase) showed a small but statistically significant increase (
1.6-fold). Both glucose-6-phosphate isomerase and fructose-diphosphate aldolase are enzymes that are active in the junction between PPP and glycolysis, and this could explain the upregulation of the genes encoding these enzymes.
S. aureus contains several genes that encode phosphoglycerate mutase. One of these genes, gpm, was highly upregulated (28.5-fold). Phosphoglycerate mutase is one of the enzymes in the glycolysis pathway. Given the high level of upregulation of this gene, a codon adaptation index (CAI) analysis was carried out (http://www.evolvingcode.net/codon/CalculateCAIs.php; University of Maryland, Baltimore County, MD) to check if gpm was a glycolytic enzyme. The CAI analysis showed that this enzyme is probably not part of the glycolysis pathway (results not shown). This was also supported by the fact that the gpm gene was not a member of the gap operon (gapA1-pgk-tpiA-pgm-eno). Further work would be needed to elucidate the significance of this upregulation.
The expression of nine genes linked to electron transport was significantly changed by the acid shock. Among these were the three thioredoxin genes (trxAB and SACOL1794) discussed above and the NADH dehydrogenase gene (nuoF), and also two upregulated NAD(P)H oxidoreductase genes (frp and SACOL0453).
Effects of acid shock on virulence and persistence factors.
The acid shock did not seem to influence the induction of toxins, since most genes encoding toxins were down-regulated (Table 3, section C3). This is in accordance with their expected initiation at post-exponential growth. The active toxin in S. aureus 50583 is toxin B. SarA has been shown to upregulate both toxic shock syndrome toxin 1 gene (tst) expression and staphylococcal enterotoxin B production (Chan & Foster, 1998). Neither the sarA gene nor the toxin B gene showed a significant change of expression level after the acid shock. The two-component system genes saeR and saeS, which are parts of the sae signalling locus involved in the regulation of many exoproteins (Novick & Jiang, 2003), were both down-regulated (saeR 0.35-fold and saeS 0.48-fold after 20 min). These results are consistent with those of Weinrick et al. (2004), which show that mild acid (3 h at pH 5.5) reduces expression of sae genes 2.1-fold. For other regulators, the results were different, e.g. in the case of the rot (repressor of toxins) locus, for which we did not observe a down-regulation of expression.
Besides the enterotoxins, a large number of other secreted proteins (haemolysins, proteases, lipases and coagulases) as well as cell-surface-associated proteins (protein A, and fibrinogen-, fibronectin- and collagen-binding proteins) are essential for S. aureus pathogenicity. With the exceptions of a putative haemolysin gene (SACOL0762) and two pathogenicity island genes (SACOL0900 and SACOL0902), most virulence genes were down-regulated or showed no statistically significant change in expression ratio.
Of the genes directly related to drug resistance and detoxification of the cell, the genes encoding a Dps family protein (SACOL2131), alkaline-shock protein 23 and three drug-transporter genes were all upregulated. The asp23 gene, encoding the alkaline shock protein 23, showed a somewhat unusual response in that it was first down-regulated and then upregulated only after 20 min. The pattern of expression remains unclear, since asp23 is reported to be under the control of B (Giachino et al., 2001), which itself showed no change in expression. The upregulation of the putative drug-resistance transporter EmrB/QacA (SACOL2347 and SACOL2348) was at an especially high level, with factors of 12.1 and 15.5, respectively (Table 3, section C2). The multidrug-resistance gene norA, the drug-transporter gene SACOL2460 and the bicyclomycin-resistance gene bcr were all down-regulated.
For the extracellular enzymes, our results showed down-regulation of staphylocoagulase precursor (SA0857), which is involved in clumping bacteria together as an antiphagocytic defence. Catalase, which protects the cells from being killed by polymorphonuclear leukocytes (PMNs), was (as mentioned above) clearly upregulated after acidification. Higher levels of catalase activity in virulent strains than in non-virulent strains of S. aureus have been found (Kanafani & Martin, 1985). Nucleases are important for virulence in that they permit the reuse of bacterial and host nucleic acids. Several nucleases were found to be upregulated (uvrC, nth, rexA, rexB), while some were down-regulated (sbcD, recJ, nfo).
The results indicated lack of adhesion and initiation of biofilm formation in the first 20 min following the acid shock. None of the major biofilm regulators sigB, agr or sarA showed a statistically significant change in expression after the acid shock. The ica genes (icaADBC), known to be central to intercellular adhesion and biofilm formation, were all strongly down-regulated to a minimum ratio of 0.12–0.15. This was in accordance with the result obtained when cells were subjected to peracetic acid (Chang et al., 2006), but in contrast to work showing upregulation of the icaA and icaD genes when cells are grown at pH 5.5 (Weinrick et al., 2004). Two other adherence-associated genes, fnbA (SACOL2511) and fnbB, encoding fibronectin-binding proteins A and B, respectively, were down-regulated. The clfA gene, encoding clumping factor A, mediates adherence to polyethylene and polyvinylchloride (Vaudaux et al., 1995), and showed a clear increase in expression from 10 to 20 min after the acid shock. The increase seen for clumping factor A could be an indication of adhesion after 20 min, but further work is necessary to confirm this. It should be added that S. aureus 50583 produces relatively little biofilm compared to other S. aureus strains at 37 °C (Rode et al., 2007).
Genes of unknown function.
Among the 622 genes that showed a statistically significant change of expression level, 29 (4.7 %) encode enzymes of unknown specificity, 38 (6.1 %) encode proteins of unknown function and 133 (21.4 %) encode hypothetical proteins. Of the genes encoding hypothetical proteins, 59 (9.5 % of 622) were up- or down-regulated more than twofold and were included in Table 3 (section J3). Further work will be required to elucidate their role in the acid response.
Conclusions
Most responses showed an increase or decrease from time zero to 10 min after acidification and then generally a stabilization in expression level from 10 to 20 min. Increased urease activity appears to be an important factor in the acid defence of S. aureus 50583, along with proton excretion by NADH dehydrogenase and macromolecule-repair mechanisms. Oxidative-stress responses, such as increased expression of thioredoxin genes and upregulation of PPP genes to generate more reducing power, were observed. A general reduction in the expression of genes encoding ribosomal proteins and genes involved in nucleotide synthesis, as well as fatty acid and lipoprotein metabolism, was typical, and reflected the lowered growth rate after acidification. Repair mechanisms and degradation of damaged proteins were induced. The acid shock did not initiate a virulence response. Many of the presented observations can be explained; some are unknown or cannot easily be interpreted. Nevertheless, together, these results show the main responses of S. aureus and will be a good starting point for future, more specific, in-depth studies of gene responses associated with the acid-stress defence of S. aureus.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: J. Green
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Armstrong-Buisseret, L., Cole, M. B. & Stewart, G. S. A. B. (1995). A homolog to the Escherichia coli alkyl hydroperoxide reductase ahpC is induced by osmotic upshock in Staphylococcus aureus. Microbiology 141, 1655–1661.
Bauerfeind, P., Garner, R. M. & Mobley, H. L. T. (1996). Allelic exchange mutagenesis of nixA in Helicobacter pylori results in reduced nickel transport and urease activity. Infect Immun 64, 2877–2880.[Abstract]
Beenken, K. E., Dunman, P. M., McAleese, F., Macapagal, D., Murphy, E., Projan, S. J., Blevins, J. S. & Smeltzer, M. S. (2004). Global gene expression in Staphylococcus aureus biofilms. J Bacteriol 186, 4665–4684.
Brown, N. L., Stoyanov, J. V., Kidd, S. P. & Hobman, J. L. (2003). The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145–163.[CrossRef][Medline]
Bunik, V. I. (2003). 2-Oxo acid dehydrogenase complexes in redox regulation – role of the lipoate residues and thioredoxin. Eur J Biochem 270, 1036–1042.[Medline]
Burne, R. A. & Chen, Y. Y. M. (2000). Bacterial ureases in infectious diseases. Microbes Infect 2, 533–542.[CrossRef][Medline]
Chan, P. F. & Foster, S. J. (1998). Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J Bacteriol 180, 6232–6241.
Chang, W., Toghrol, F. & Bentley, W. E. (2006). Toxicogenomic response of Staphylococcus aureus to peracetic acid. Environ Sci Technol 40, 5124–5131.[Medline]
Chastanet, A., Derre, I., Nair, S. & Msadek, T. (2004). clpB, a novel member of the Listeria monocytogenes CtsR regulon, is involved in virulence but not in general stress tolerance. J Bacteriol 186, 1165–1174.
Chatterjee, I., Becker, P., Grundmeier, M., Bischoff, M., Somerville, G. A., Peters, G., Sinha, B., Harraghy, N., Proctor, R. A. & Herrmann, M. (2005). Staphylococcus aureus ClpC is required for stress resistance, aconitase activity, growth recovery, and death. J Bacteriol 187, 4488–4496.
Chedin, F. & Kowalczykowski, S. C. (2002). A novel family of regulated helicases/nucleases from Gram-positive bacteria: insights into the initiation of DNA recombination. Mol Microbiol 43, 823–834.[CrossRef][Medline]
Chen, Y. Y. M., Weaver, C. A., Mendelsohn, D. R. & Burne, R. A. (1998). Transcriptional regulation of the Streptococcus salivarius 57.I urease operon. J Bacteriol 180, 5769–5775.
Clements, M. O. & Foster, S. J. (1999). Stress resistance in Staphylococcus aureus. Trends Microbiol 7, 458–462.[CrossRef][Medline]
Clements, M. O., Watson, S. P. & Foster, S. J. (1999). Characterization of the major superoxide dismutase of Staphylococcus aureus and its role in starvation survival, stress resistance, and pathogenicity. J Bacteriol 181, 3898–3903.
Cohen, S. P., Hachler, H. & Levy, S. B. (1993). Genetic and functional analysis of the multiple antibiotic-resistance (mar) locus in Escherichia coli. J Bacteriol 175, 1484–1492.
Cotter, P. D. & Hill, C. (2003). Surviving the acid test: responses of Gram-positive bacteria to low pH. Microbiol Mol Biol Rev 67, 429–453.
Davis, G. S. & Mobley, H. L. T. (2005). Contribution of dppA to urease activity in Helicobacter pylori 26695. Helicobacter 10, 416–423.[CrossRef][Medline]
Deora, R., Tseng, T. & Misra, T. K. (1997). Alternative transcription factor SB of Staphylococcus aureus: characterization and role in transcription of the global regulatory locus sar. J Bacteriol 179, 6355–6359.
Epstein, W. (2003). The roles and regulation of potassium in bacteria. Prog Nucleic Acid Res Mol Biol 75, 293–320.[Medline]
Foster, J. W. (2004). Escherichia coli acid resistance: tales of an amateur acidophile. Nat Rev Microbiol 2, 898[CrossRef][Medline]
Frees, D., Chastanet, A., Qazi, S., Sorensen, K., Hill, P., Msadek, T. & Ingmer, H. (2004). Clp ATPases are required for stress tolerance, intracellular replication and biofilm formation in Staphylococcus aureus. Mol Microbiol 54, 1445–1462.[CrossRef][Medline]
Giachino, P., Engelmann, S. & Bischoff, M. (2001). B activity depends on RsbU in Staphylococcus aureus. J Bacteriol 183, 1843–1852.
Greenberg, J. T., Monach, P., Chou, J. H., Josephy, P. D. & Demple, B. (1990). Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci U S A 87, 6181–6185.
Hofmann, B., Hecht, H. J. & Flohe, L. (2002). Peroxiredoxins. Biol Chem 383, 347–364.[CrossRef][Medline]
Horsburgh, M. J., Clements, M. O., Crossley, H., Ingham, E. & Foster, S. J. (2001). PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69, 3744–3754.
Kanafani, H. & Martin, S. E. (1985). Catalase and superoxide-dismutase activities in virulent and nonvirulent Staphylococcus aureus isolates. J Clin Microbiol 21, 607–610.
Kisker, C., Schindelin, H. & Rees, D. C. (1997). Molybdenum-cofactor-containing enzymes: structure and mechanism. Annu Rev Biochem 66, 233–267.[CrossRef][Medline]
Kobayashi, H., Miyamoto, T., Hashimoto, Y., Kiriki, M., Motomatsu, A., Honjoh, K. & Iio, M. (2005). Identification of factors involved in recovery of heat-injured Salmonella Enteritidis. J Food Prot 68, 932–941.[Medline]
Kuroda, M., Kuroda, H., Oshima, T., Takeuchi, F., Mori, H. & Hiramatsu, K. (2003). Two-component system VraSR positively modulates the regulation of cell-wall biosynthesis pathway in Staphylococcus aureus. Mol Microbiol 49, 807–821.[CrossRef][Medline]
Langsrud, O. (2005). Rotation tests. Stat Comput 15, 53–60.[CrossRef]
Lasken, R. S. & Kornberg, A. (1988). The primosomal protein N' of Escherichia coli is a DNA helicase. J Biol Chem 263, 5512–5518.
Len, A. C., Harty, D. W. S. & Jacques, N. A. (2004). Proteome analysis of Streptococcus mutans metabolic phenotype during acid tolerance. Microbiology 150, 1353–1366.
Lowy, F. D. (1998). Medical progress – Staphylococcus aureus infections. N Engl J Med 339, 520–532.
Ma, J. F., Hager, P. W., Howell, M. L., Phibbs, P. V. & Hassett, D. J. (1998). Cloning and characterization of the Pseudomonas aeruginosa zwf gene encoding glucose-6-phosphate dehydrogenase, an enzyme important in resistance to methyl viologen (paraquat). J Bacteriol 180, 1741–1749.
Manna, A. C., Bayer, M. G. & Cheung, A. L. (1998). Transcriptional analysis of different promoters in the sar locus in Staphylococcus aureus. J Bacteriol 180, 3828–3836.
Maurer, L. M., Yohannes, E., Bondurant, S. S., Radmacher, M. & Slonczewski, J. L. (2005). pH regulates genes for flagellar motility, catabolism, and oxidative stress in Escherichia coli K-12. J Bacteriol 187, 304–319.
Miyazaki, E., Chen, J. M., Ko, C. & Bishai, W. R. (1999). The Staphylococcus aureus rsbW (orf159) gene encodes an anti-sigma factor of SigB. J Bacteriol 181, 2846–2851.
Mobley, H. L. T., Garner, R. M. & Bauerfeind, P. (1995). Helicobacter-pylori nickel-transport gene nixA – synthesis of catalytically active urease in Escherichia coli independent of growth-conditions. Mol Microbiol 16, 97–109.[Medline]
Moen, B., Oust, A., Langsrud, O., Dorrell, N., Marsden, G. L., Hinds, J., Kohler, A., Wren, B. W. & Rudi, K. (2005). Explorative multifactor approach for investigating global survival mechanisms of Campylobacter jejuni under environmental conditions. Appl Environ Microbiol 71, 2086–2094.
Novick, R. P. & Jiang, D. R. (2003). The staphylococcal saeRS system coordinates environmental signals with agr quorum sensing. Microbiology 149, 2709–2717.
Nurse, P., Zavitz, K. H. & Marians, K. J. (1991). Inactivation of the Escherichia coli PriA DNA-replication protein induces the SOS response. J Bacteriol 173, 6686–6693.
Paget, M. S. B., Molle, V., Cohen, G., Aharonowitz, Y. & Buttner, M. J. (2001). Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the R regulon. Mol Microbiol 42, 1007–1020.[CrossRef][Medline]
Pallen, M. J. & Wren, B. W. (1997). The HtrA family of serine proteases. Mol Microbiol 26, 209–221.[CrossRef][Medline]
Palma, M. & Cheung, A. L. (2001). B activity in Staphylococcus aureus is controlled by RsbU and an additional factor(s) during bacterial growth. Infect Immun 69, 7858–7865.
Quiberoni, A., Rezaiki, L., El Karoui, M., Biswas, I., Tailliez, P. & Gruss, A. (2001). Distinctive features of homologous recombination in an ‘old’ microorganism, Lactococcus lactis. Res Microbiol 152, 131–139.[Medline]
Rode, T. M., Langsrud, S., Holck, A. & Møretrø, T. (2007). Different patterns of biofilm formation in Staphylococcus aureus under food-related stress conditions. Int J Food Microbiol 116, 372–383.[CrossRef][Medline]
Tsau, J. L., Guffanti, A. A. & Montville, T. J. (1992). Conversion of pyruvate to acetoin helps to maintain pH homeostasis in Lactobacillus plantarum. Appl Environ Microbiol 58, 891–894.
Uziel, O., Borovok, I., Schreiber, R., Cohen, G. & Aharonowitz, Y. (2004). Transcriptional regulation of the Staphylococcus aureus thioredoxin and thioredoxin reductase genes in response to oxygen and disulfide stress. J Bacteriol 186, 326–334.
Valenzuela, M., Cerda, O. & Toledo, H. (2003). Overview on chemotaxis and acid resistance in Helicobacter pylori. Biol Res 36, 429–436.[Medline]
van Vliet, A. H. M., Kuipers, E. J., Waidner, B., Davies, B. J., de Vries, N., Penn, C. W., Vandenbroucke-Grauls, C. M., Kist, M., Bereswill, S. & Kusters, J. G. (2001). Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level. Infect Immun 69, 4891–4897.
Vaudaux, P. E., Francois, P., Proctor, R. A., McDevitt, D., Foster, T. J., Albrecht, R. M., Lew, D. P., Wabers, H. & Cooper, S. L. (1995). Use of adhesion-defective mutants of Staphylococcus aureus to define the role of specific plasma proteins in promoting bacterial adhesion to canine arteriovenous shunts. Infect Immun 63, 585–590.[Abstract]
Weinrick, B., Dunman, P. M., McAleese, F., Murphy, E., Projan, S. J., Fang, Y. & Novick, R. P. (2004). Effect of mild acid on gene expression in Staphylococcus aureus. J Bacteriol 186, 8407–8423.
Wilkins, J. C., Homer, K. A. & Beighton, D. (2002). Analysis of Streptococcus mutans proteins modulated by culture under acidic conditions. Appl Environ Microbiol 68, 2382–2390.
Wood, J. M. (1999). Osmosensing by bacteria: signals and membrane-based sensors. Microbiol Mol Biol Rev 63, 230–262.
Received 10 January 2007;
revised 16 March 2007;
accepted 19 March 2007.
This article has been cited by other articles:
|
|
 |
|
  S. Even, C. Charlier, S. Nouaille, N. L. Ben Zakour, M. Cretenet, F. J. Cousin, M. Gautier, M. Cocaign-Bousquet, P. Loubiere, and Y. Le Loir Staphylococcus aureus Virulence Expression Is Impaired by Lactococcus lactis in Mixed Cultures Appl. Envir. Microbiol., July 1, 2009; 75(13): 4459 - 4472. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. C. Wilks, R. D. Kitko, S. H. Cleeton, G. E. Lee, C. S. Ugwu, B. D. Jones, S. S. BonDurant, and J. L. Slonczewski Acid and Base Stress and Transcriptomic Responses in Bacillus subtilis Appl. Envir. Microbiol., February 15, 2009; 75(4): 981 - 990. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schlag, S. Fuchs, C. Nerz, R. Gaupp, S. Engelmann, M. Liebeke, M. Lalk, M. Hecker, and F. Gotz Characterization of the Oxygen-Responsive NreABC Regulon of Staphylococcus aureus J. Bacteriol., December 1, 2008; 190(23): 7847 - 7858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Raafat, K. von Bargen, A. Haas, and H.-G. Sahl Insights into the Mode of Action of Chitosan as an Antibacterial Compound Appl. Envir. Microbiol., June 15, 2008; 74(12): 3764 - 3773. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Reid, R. Pandey, K. Palyada, H. Naikare, and A. Stintzi Identification of Campylobacter jejuni Genes Involved in the Response to Acidic pH and Stomach Transit Appl. Envir. Microbiol., March 1, 2008; 74(5): 1583 - 1597. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-C. Yuan, P. Liu, P. Saenkham, K. Kerr, and E. W. Nester Transcriptome Profiling and Functional Analysis of Agrobacterium tumefaciens Reveals a General Conserved Response to Acidic Conditions (pH 5.5) and a Complex Acid-Mediated Signaling Involved in Agrobacterium-Plant Interactions J. Bacteriol., January 15, 2008; 190(2): 494 - 507. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, Acidified cells; 
, control without acid addition. The results are the means of four experiments; error bars represent SD.