The experiences of internationally educated nurses in the southeastern United States of America.

BACKGROUND: US healthcare facilities have addressed nursing shortages in part by recruiting internationally educated nurses (IENs), and studies suggest IENs may make up a significant percentage of the nursing workforce in urban hospitals. Despite the economic recession of 2008-2012, international nurse migration is expected to continue. Little is known about IENs in the southeastern USA, and no studies have compared their perspectives to those of their US counterparts. OBJECTIVE: The purpose of this study was to gain a deeper understanding about the experiences of IENs compared to those of US registered nurses (RNs) practising in two urban hospitals in southeastern USA. METHODS: This study involved two rounds of semi-structured interviews of 82 IENs and US RNs. Interviews focused on themes relating to education, barriers to practice, intent to stay in nursing and IENs' migration experiences. FINDINGS AND DISCUSSION: Most IENs interviewed migrated to the USA after 1990 to join their family and do not plan to return to their home countries to practise. Most IENs initially received their Associate Degree in Nursing; many have obtained their Bachelor of Science in Nursing degree. IENs and newly licensed US RNs faced similar barriers when they began practising in the USA, but IENs faced additional challenges adjusting to the attitudes of US patients, the perceived lack of respect for nurses and delivering total patient care. CONCLUSIONS: IENs would benefit from orientation regarding the cultural differences in the USA. In other ways, their challenges are similar to those of US RNs; policies regarding education, recruitment and retention could target both groups together.

ClpP is involved in the stress response and degradation of misfolded proteins in Salmonella enterica serovar Typhimurium.

Components of the ATP-dependent Clp protease complex are found in a wide range of prokaryotic cells and they are often expressed as part of the cellular stress response. To investigate the physiological role of the proteolytic subunit, ClpP, in Salmonella enterica serovar Typhimurium (S. typhimurium) an in-frame deletion of the clpP gene was constructed. Growth experiments revealed that clpP is important for the ability of S. typhimurium to grow under various stressful conditions, such as low pH, elevated temperature and high salt concentrations. Since the stationary-phase sigma factor, RpoS, is a target of the Clp proteolytic complex, the effect of the clpP deletion in the absence of RpoS was examined; it was observed that growth of the S. typhimurium clpP mutant is affected in both an RpoS-dependent and an RpoS-independent manner. Analysis of the degradation of abnormal puromycyl-containing polypeptides showed that ClpP participates in the proteolysis of such proteins in S. typhimurium. These findings prompted an investigation of the growth of an Escherichia coli clpP mutant under various stress conditions. The growth of this E. coli mutant was affected by heat, salt and low pH, although not to the same extent as observed for the S. typhimurium clpP mutant. The results of this study indicate that the S. typhimurium clpP mutant is generally more sensitive to environmental stress than the E. coli clpP mutant and it is proposed that this is due to a reduced ability to degrade misfolded proteins generated under these conditions.

An activator of glutamate decarboxylase genes regulates the expression of enteropathogenic Escherichia coli virulence genes through control of the plasmid-encoded regulator, Per.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhoea in a number of developing countries and is the prototype of pathogenic bacteria that cause attaching and effacing (A/E) intestinal lesions. A chromosomal pathogenicity island, termed the locus of enterocyte effacement (LEE), contains all the genes necessary for the A/E phenotype as well as genes for a type III secretion system and intimate adhesion. Genes in the LEE and genes involved in the synthesis of bundle-forming pili (BFP) are positively regulated by the plasmid-encoded regulator (Per) and comprise the per regulon. In order to identify factors that control the per regulon, we screened an EPEC genomic library for clones that modulate the expression of per. A plasmid clone that decreased the expression of per was isolated using a lacZ reporter gene fused to the per promoter. Subcloning revealed that YhiX, a putative AraC/XylR family transcriptional regulator, was the effector of per repression. Through downregulation of per, a plasmid overproducing YhiX reduced the synthesis of intimin, BfpA, Tir, and CesT, factors important for EPEC virulence. yhiX is located downstream of gadA, which encodes glutamate decarboxylase, an enzyme involved in acid resistance of E. coli. YhiX was found to be an activator of gadA, and the cloned yhiX gene increased production of glutamate decarboxylases (GAD) and activated the transcription of the gadA and gadB promoters. Therefore, yhiX was renamed gadX. Analysis of a gadX mutant grown in the different culture media with acidic and alkaline pH showed that regulation of perA, gadA and gadB by GadX was altered by the external pH and the culture media condition. Under conditions in which EPEC infects cultured epithelial cells, GadX negatively regulated perA expression, and the derepression in the gadX mutant increased translocation of Tir into epithelial cells relative to wild-type EPEC. DNA mobility shift experiments showed that purified GadX protein bound to the perA, gadA and gadB promoter regions in vitro, indicating that GadX is a transcriptional regulator of these genes. On the basis of these results, we propose that GadX may be involved in the appropriate expression of genes required for acid resistance and virulence of EPEC. Our data are consistent with a model in which environmental changes resulting from passage from the stomach to the proximal small intestine induce the functional effect of GadX on per and GAD expression in order to prevent inappropriate expression of the products of these two systems.

Breaking through the acid barrier: an orchestrated response to proton stress by enteric bacteria.

The ability of enteropathogens such as Salmonella and Escherichia coli to adapt and survive acid stress is fundamental to their pathogenesis. Once inside the host, these organisms encounter life-threatening levels of inorganic acid (H+) in the stomach and a combination of inorganic and organic acids (volatile fatty acids) in the small intestine. To combat these stresses, enteric bacteria have evolved elegant, overlapping strategies that involve both constitutive and inducible defense systems. This article reviews the recent progress made in understanding the pH 3 acid tolerance systems of Salmonella and the even more effective pH 2 acid resistance systems of E. coli. Focus is placed on how Salmonella orchestrates acid tolerance by modulating the activities or levels of diverse regulatory proteins in response to pH stress. The result is induction of overlapping arrays of acid shock proteins that protect the cell against acid and other environmental stresses. Most notable among these pH-response regulators are RpoS, Fur, PhoP and OmpR. In addition, we will review three dedicated acid resistance systems of E. coli, not present in Salmonella, that allow this organism to survive extreme (pH 2) acid challenge.

Spiroplasma sp. 16S rDNA in Creutzfeldt-Jakob disease and scrapie as shown by PCR and DNA sequence analysis.

The pathogenesis of the transmissible spongiform encephalopathies (TSE), which include Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep, remains an enigma. In this paper we present evidence for the association of Spiroplasma sp., a wall-less prokaryote, with TSE. We have shown PCR amplification of Spiroplasma 16S rDNA in TSE-infected brain tissues (13 of 13 CJD cases and 5 of 9 scrapie cases) and not in control brains (0 of 50). Direct sequencing of the amplified PCR products has confirmed the presence of Spiroplasma-like DNA in all 5 of the TSE brains tested. Our evidence is not necessarily in conflict with involvement of a PrPres--a protease-resistant host-derived protein referred to as the prion--in the pathogenesis of TSE, since there is evidence that another factor is involved. We propose a bacterium, namely Spiroplasma, as this associated factor although the role of Spiroplasma in TSE cannot be determined from these experiments. The presence of the nucleic acid sequence of this microbe in all cases of TSE in our laboratory and not in controls provides direct evidence of the association of Spiroplasma sp. with TSE.

Regulation of sigma S degradation in Salmonella enterica var typhimurium: in vivo interactions between sigma S, the response regulator MviA(RssB) and ClpX.

The alternate sigma factor sigmaS plays an important role in the survival of Salmonella typhimurium following sudden encounters with a variety of stress conditions. The level of sigmaS is very low in rapidly growing cells but dramatically increases as those cells encounter environmental stress or enter into stationary phase. This increase is due in large measure to the stabilization of sigmaS protein against degradation by the ClpXP protease. The MviA protein, also known as RssB or SprE in Escherichia coli, is a putative member of a two component signal transduction system that plays a central role in facilitating sigmaS degradation by ClpXP. In contrast to most two-component systems, MviA does not appear to regulate gene expression but is believed to interact directly with sigmaS and somehow facilitate degradation. We now provide evidence that MviA(RssB) directly interacts both with sigmaS and ClpX in vivo, presumably enabling presentation of sigmaS to the ClpP protease. Interactions were demonstrated using a bacterial two-hybrid system in which sigmaS, MviA, and ClpX were fused to separate moieties of Bordetella pertussis CyaA (adenylate cyclase). Paired hybrid plasmids containing Cya'-MviA/RpoS-'Cya or Cya'-MviA/ClpX-'Cya successfully reconstituted adenylate cyclase activity in both S. typhimurium and E. coli. However, no direct interactions were detected between ClpX and RpoS. A second series of experiments has indicated that the interaction between MviA and sigmaS requires the N-terminus but not the C-terminus of MviA. Cellular levels of MviA appear to be very low in the cell based on lacZ fusion, Western blot and Northern blot analyses suggesting a catalytic role for MviA in sigmaS degradation. Mutagenesis of MviA residue D58, a canonical residue subject to phosphorylation in many two-component systems, decreased the ability of MviA to facilitate sigmaS turnover in vivo confirming that phosphorylation of MviA increases MviA activity.

Field anesthesia of free-living mountain gorillas (Gorilla gorilla beringei) from the Virunga Volcano region, Central Africa.

Twenty-six anesthetic procedures involving 24 free-living mountain gorillas (Gorilla gorilla beringei) from Rwanda or the Democratic Republic of Congo were performed between February 1987 and October 1997. Sixteen procedures were performed to remove snares or to treat snare-related wounds, and four of the animals died without recovering consciousness because of their severe medical conditions. Ketamine was used for induction 19 times, tiletamine/zolazepam was used five times, and the agent was not recorded for two procedures. The mean (+/- SD) ketamine dosage for four animals of known weight was 7.1 +/- 0.9 mg/kg. All induction agents were delivered i.m. by remote injection, and mean induction times for ketamine and tiletamine/zolazepam were 5.5 +/- 2.6 min (n = 12) and 5.4 +/- 3.7 min (n = 5), respectively. Mean recovery times were significantly shorter with ketamine compared with tiletamine/zolazepam (42.0 +/- 24.9 min, n = 9 vs. 75.25 +/- 22.1 min, n = 4). Low hemoglobin oxygen saturation (mean = 86.7%) was recorded in three cases under ketamine anesthesia, and oxygen insufflation is therefore recommended to prevent hypoxemia. Gorillas induced with tiletamine/zolazepam had significantly higher respiratory rates compared with animals given ketamine. Successful anesthesia and recovery, in particular, depended on the assistance of local personnel.

OmpR regulates the stationary-phase acid tolerance response of Salmonella enterica serovar typhimurium.

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria. Salmonella enterica serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422-1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene ompR. The ompR mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR, ompC and ompF, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of ompC. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an ack pta mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of ompR message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.

Adaptive response to cold temperatures and characterization of cspA in Salmonella typhimurium LT2.

Salmonella typhimurium is a major foodborne microbial pathogen which primarily contaminates poultry products causing salmonellosis in humans. S. typhimurium LT2 cultures, when transferred from 37 degrees C to 5 degrees C or 10 degrees C, showed an initial lag period in growth with an approximate generation time of 10-25 h. Western blot assay using E. coli CS7.4 antibody and analysis of radiolabeled total cellular proteins from S. typhimurium cultures after exposure to 10 degrees C or 5 degrees C showed elevated expression of a major cold shock protein, CS7.4. Identification of a decreased level of CS7.4 at 37 degrees C suggests that the expression of this protein may require a large temperature downshift. Putative regulatory protein binding segment on the 5'-untranslated region referred as 'Fragment 7' in S. typhimurium exhibited a 90.6% and a 56.25% nucleotide sequence identity when compared with the Fragment 7 of E. coli and S. enteritidis, respectively. The differences in the nucleotide sequence within the Fragment 7 between S. typhimurium and S. enteritidis may explain the differential expression of CspA at 37 degrees C. The nucleotide sequence of the open reading frame of S. typhimurium cspA gene showed a single base difference at 816 bp position from a G to a C which altered the amino acid residue from a glycine to an alanine. In addition to CspA, an elevated expression of a 105 kDa, and decreased expression of 6 proteins were evidenced when cultures of S. typhimurium were exposed to 10 degrees C or 5 degrees C. Differential expression of the CspA and other proteins in S. typhimurium following exposure to cold temperatures suggest that adaptation and continued growth and survival at cold temperatures in this pathogen may be aided by these cold-responsive proteins.

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7.

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P

Gastrointestinal parasites of mountain gorillas (Gorilla gorilla beringei) in the Parc National des Volcans, Rwanda.

Ninety-eight fecal samples were collected from 74 free-living mountain gorillas (Gorilla gorilla beringei) from the Parc National des Volcans, Rwanda, between July 1995 and January 1997 and examined for parasites by Sheather's sugar and zinc sulfate flotation methods, trichrome staining, and larval cultures. All samples contained at least one parasite. Seventeen endoparasites were identified, including eight protozoa, seven nematodes, one cestode, and one trematode. Two species of arthropod mite were also recovered from the fecal samples. Parasites observed on fecal examinations included strongyle/trichostrongyle-type eggs (72/74) (representing Oesphagostomum sp., Trichostrongylus sp., Hyostrongylus spp., and possibly Murshidia sp.), Strongyloides sp. (1/74), Trichuris trichiura (2/74), Probstmayria sp. (7/74), Anoplocephala sp. (63/74), Entamoeba hartmanni cysts and trophozoites (19/70), Endolimax nana cysts (31/70), Iodamoeba buetschlii cysts (11/70), Endolimax nana or Iodamoeba buetschlii trophozoites (63/70). Entamoeba coli cysts and trophozoites (14/70), Entamoeba histolytica trophozoite (1/70), Chilomastix sp. cysts and trophozoites (31/70), and Giardia sp. cysts (2/70). In addition, one ascarid and one trematode egg were seen. There were no significant differences in the prevalence of parasites between males and females and between age groups: however, infants and juveniles appeared to have a lower prevalence of Anoplocephala gorillae, and the silverbacked males appeared to have a higher prevalence of Probstmayria sp. Parasite prevalence was consistent among the five social groups studied except Susa group had a significantly lower prevalence of Anoplocephala gorillae. Trichuris trichiura, Strongyloides sp., Chilomastix sp., and Endolimax nana were identified for the first time in this population, and it is possible that these parasites were of human origin. Although there were no obvious clinical effects due to the presence of these parasites, six parasites identified (Trichuris trichiura, Strongyloides sp., Oesphagostomum sp., Trichostrongylus sp., Entamoeba histolytica, and Giardia sp.) could potentially be pathogenic. Some of the parasite products and cultured larvae could not be speciated.

Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium.

Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor sigmaS. The repertoire of sigmaS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering sigmaS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating sigmaS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading sigmaS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating sigmaS levels. As predicted, clpP mutants accumulated large amounts of sigmaS even in log phase. However, dksA mutants failed to accumulate sigmaS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS-PAGE analysis of whole-cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for sigmaS in pathogenesis.

Virulent Salmonella typhimurium has two periplasmic Cu, Zn-superoxide dismutases.

Periplasmic Cu, Zn-cofactored superoxide dismutase (SodC) protects Gram-negative bacteria from exogenous oxidative damage. The virulent Salmonella typhimurium strain ATCC 14028s has been found to contain two discrete periplasmic Cu, Zn-SOD enzymes that are only 57% identical at the amino acid level. SodCI is carried by a cryptic bacteriophage, and SodCII is closely related to the Cu, Zn-superoxide dismutase of Escherichia coli. All Salmonella serotypes appear to carry the sodCII locus, but the phage-associated sodCI gene is found only in certain strains belonging to the most highly pathogenic serotypes. Expression of either sodC locus appears to be enhanced during stationary phase, but only sodCII is regulated by the alternative sigma factor sigmas (RpoS). Mutants lacking both sodC genes are less lethal for mice than mutants possessing either sodC locus alone, indicating that both Cu, Zn-SOD enzymes contribute to Salmonella pathogenicity. The evolutionary acquisition of an additional sodC gene has contributed to the enhanced virulence of selected Salmonella strains.

Control of acid resistance in Escherichia coli.

Acid resistance (AR) in Escherichia coli is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in E. coli. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (adiA), and the regulator cysB, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (gadA or gadB), and the gene encoding the putative glutamate:gamma-aminobutyric acid antiporter (gadC). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the gad genes proved separable. Stationary-phase induction of gadA and gadB required the alternative sigma factor sigmaS encoded by rpoS. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be sigmaS independent. Neither gad gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the sigmaS-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus, E. coli possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.

When protons attack: microbial strategies of acid adaptation.

Inducible tolerance to acidic and alkaline environments is recognized as an important survival strategy for many prokaryotic and eukaryotic microorganisms. Recent developments in understanding this phenomenon include the identification of regulatory genes, specific tolerance mechanisms and genes associated with tolerance. In addition, there is significant evidence linking pH responses with virulence.

Inducible acid tolerance mechanisms in enteric bacteria.

Enteric micro-organisms have developed several inducible mechanisms for surviving transient periods of extreme acid stress. Salmonella typhimurium possesses an acid tolerance response (ATR) induced in minimal medium by short exposures to mild acid stress. More than 50 acid shock proteins (ASPs) are induced during adaptation. Eight ASPs are regulated by the major iron regulatory protein, Fur, in an unusual iron-independent manner. The two-component regulator, PhoP, is an autoinduced ASP that controls the induction of three additional ASPs. The stress sigma factor sigma S is an ASP that regulates induction of eight ASPs. Acid induction of sigma S is due to its decreased proteolytic turnover via the ClpXP protease in conjunction with the two-component-type response regulator MviA (RssB in Escherichia coli). Mutations in any of these three regulators leads to a defective ATR. Repair of pH stress-induced DNA damage appears to require the Ada protein (O6-methylguanine methyltransferase) since an ada mutant is both acid and alkaline sensitive. In contrast to S. typhimurium, E. coli and Shigella have acid resistance systems induced in complex media that include a glucose-repressed system protective at pH 2.5 without amino acid supplementation, a glutamate decarboxylase system that requires glutamate and an arginine decarboxylase system unique to E. coli.

Adaptive response to cold temperatures in Vibrio vulnificus.

The effectiveness of rapid chilling or freezing of oysters to reduce Vibrio vulnificus levels in shellfish may be compromised by product handling procedures that permit cold adaptation. When a V. vulnificus culture was shifted from 35 degrees C to 6 degrees C conditions, it underwent transition to a non-culturable state. Cells adapted to 15 degrees C prior to change to 6 degrees C condition, however, remain viable and culturable. In addition, cultures adapted to 15 degrees C were able to survive better upon freezing at -78 degrees C compared with cultures frozen directly from 35 degrees C. Inhibition of protein synthesis by addition of chloramphenicol in a V. vulnificus culture immediately prior to the exposure to the adaptive temperature eliminated inducible cold tolerance. These results suggest that cold-adaptive "protective" proteins may enhance survival and tolerance at cold temperatures. In addition, removal of iron from the growth medium by adding 2,2'-Dipyridyl prior to cold adaptation decreased the viability by approximately 2 logarithm levels. This suggests that iron plays an important role in adaptation at cold temperatures. Analysis of total cellular proteins on an SDS polyacrylamide gel electrophoresis, labeled with 35S-methionine during exposure at 15 degrees C, showed elevated expressions of a 6-kDa and a 40-kDa protein and decreased expression of an 80-kDa protein. These results suggest that, for V. vulnificus, survival and tolerance at cold temperatures could be due to the expression of cold-adaptive proteins other than previously documented major cold shock proteins such as CS7.4 and CsdA. In this study, for the first time we have shown that exposure to an intermediate cold temperature (15 degrees C) causes a cold adaptive response, helping this pathogen remain in culturable state when exposed to a much colder temperature (6 degrees C). This adaptive nature to cold temperatures could be important for shellfish industry efforts to reduce the risk of V. vulnificus infection from consuming raw oysters.

NAD-dependent DNA-binding activity of the bifunctional NadR regulator of Salmonella typhimurium.

NadR is a 45-kDa bifunctional regulator protein. In vivo genetic studies indicate that NadR represses three genes involved in the biosynthesis of NAD. It also participates with an integral membrane protein (PnuC) in the import of nicotinamide mononucleotide, an NAD precursor. NadR was overexpressed and purified as a His-tagged fusion in order to study its DNA-binding properties. The protein bound to DNA fragments containing NAD box consensus sequences. NAD proved to be the relevant in vivo corepressor, but full NAD dependence of repressor activity required nucleotide triphosphates. DNA footprint analysis and gel shift assays suggest that NadR binds as a multimer to adjacent NAD boxes. The DNA-repressor complex would sequester a potential RNA polymerase binding site and thereby decrease expression of the nad regulon.

Cyclic AMP receptor protein and TyrR are required for acid pH and anaerobic induction of hyaB and aniC in Salmonella typhimurium.

Two acid-inducible genes, aniC and aciK, that require anaerobiosis and tyrosine for expression were identified as orf326a encoding a potential amino acid/polyamine antiporter and hyaB encoding hydrogenase I, respectively. Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.

A low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella typhimurium against inorganic acid stress.

The acid tolerance response enables Salmonella typhimurium to survive exposures to potentially lethal acidic environments. The acid stress imposed in a typical assay for acid tolerance (log-phase cells in minimal glucose medium) was shown to comprise both inorganic (i.e., low pH) and organic acid components. A gene previously determined to affect acid tolerance, atbR, was identified as pgi, the gene encoding phosphoglucoisomerase. Mutations in pgi were shown to increase acid tolerance by preventing the synthesis of organic acids. Protocols designed to separate the stresses of inorganic from organic acids revealed that the regulators sigma38 (RpoS), Fur, and Ada have major effects on tolerance to organic acid stress but only minor effects on inorganic acid stress. In contrast, the two-component regulatory system PhoP (identified as acid shock protein ASP29) and PhoQ proved to be important for tolerance to inorganic [corrected] acid stress but had little effect against organic acid stress. PhoP mutants also failed to induce four ASPs, confirming a role for this regulator in acid tolerance. Acid shock induction of PhoP appears to occur at the transcriptional level and requires the PhoPQ system. Furthermore, induction by acid occurs even in the presence of high concentrations of magnesium, the ion known to be sensed by PhoQ. These results suggest that PhoQ can sense both Mg2+ and pH. Since phoP mutants are avirulent, the low pH activation of this system has important implications concerning the pathogenesis of S. typhimurium. The involvement of four regulators, two of which are implicated in virulence, underscores the complexity of the acid tolerance stress response and further suggests that features of acid tolerance and virulence are interwoven.