Characterization of Two Novel AmpC Beta-Lactamases from the Emerging Opportunistic Pathogen, Cedecea neteri.

The genus Cedecea (family Enterobacteriaceae) causes a wide spectrum of acute infections in immunocompromised hosts, from pneumonia and bacteremia to oral ulcers and dialysis-related peritonitis. While Cedecea infections are reported infrequently in the literature, documented clinical cases of this emerging opportunistic human pathogen have occurred worldwide. Cedecea neteri has clinical significance and exhibits antimicrobial drug resistance. However, little is known about the molecular basis underlying the resistance phenotypes in C. neteri. We previously hypothesized that the open-reading frame cnt10470 in the C. neteri SSMD04 genome encodes a chromosomal Ambler class C (AmpC) ß-lactamase based on sequence homology. In this study, recombinant polyhistidine-tagged proteins were created by cloning the putative ampC genes from SSMD04 and C. neteri ATCC 33855 (a clinical isolate) into the pET-6xHN expression vector, overexpressing the proteins, and then purifying the recombinant AmpCs (rAmpCs) using immobilized metal affinity chromatography (Ni-NTA). The in vitro enzymatic analysis of the purified rAmpCs was performed to determine the Km and kcat for various ß-lactam substrates. The rAmpCs are functional class C ß-lactamases when assayed using the chromogenic ß-lactamase substrate, nitrocefin. The presence of functional AmpCs in both C. neteri strains underscores the necessity of performing antibiotic susceptibility testing in the management of C. neteri infections.

Rare Heterogeneous Adverse Events Associated with mRNA-Based COVID-19 Vaccines: A Systematic Review.

Background: Since the successful development, approval, and administration of vaccines against SARS-CoV-2, the causative agent of COVID-19, there have been reports in the published literature, passive surveillance systems, and other pharmacovigilance platforms of a broad spectrum of adverse events following COVID-19 vaccination. A comprehensive review of the more serious adverse events associated with the Pfizer-BioNTech and Moderna mRNA vaccines is warranted, given the massive number of vaccine doses administered worldwide and the novel mechanism of action of these mRNA vaccines in the healthcare industry. Methods: A systematic review of the literature was conducted to identify relevant studies that have reported mRNA COVID-19 vaccine-related adverse events. Results: Serious and severe adverse events following mRNA COVID-19 vaccinations are rare. While a definitive causal relationship was not established in most cases, important adverse events associated with post-vaccination included rare and non-fatal myocarditis and pericarditis in younger vaccine recipients, thrombocytopenia, neurological effects such as seizures and orofacial events, skin reactions, and allergic hypersensitivities. Conclusions: As a relatively new set of vaccines already administered to billions of people, COVID-19 mRNA-based vaccines are generally safe and efficacious. Further studies on long-term adverse events and other unpredictable reactions in close proximity to mRNA vaccination are required.

Antibiotic resistance of Escherichia coli urinary tract infections at a North Carolina community hospital: Comparison of rural and urban community type.

OBJECTIVE: We aimed to identify differences in urinary E. coli resistance rates based on community type of patient residence (rural and urban). METHODS: This cross-sectional study examined antibiotic resistance of E. coli isolates from 12,604 urine specimens at a North Carolina hospital between 2016 and 2018. Using multivariable logistic regression modeling, we investigated the association between resistance and community type, adjusting for patient age, gender, season, and setting of infection onset. Analyses were performed using SAS Version 9.3 (SAS Institute, Cary, NC) at alpha = 0.05. RESULTS: Prevalence of resistance was highest for ampicillin (42.2%), ampicillin-sulbactam (24.7%), ciprofloxacin (21.8%), trimethoprim-sulfamethoxazole (SXT) (21.6%), and levofloxacin (21.4%). Rural compared to urban community type was significantly associated with resistance of E. coli urinary isolates to ciprofloxacin (adjusted odds ratio [aOR] = 1.29, 95% confidence interval [CI] = 1.16-1.43, P < .0001), levofloxacin (aOR = 1.28, 95% CI = 1.15-1.42, P < .0001), SXT (aOR = 1.15, 95% CI = 1.04-1.27, P = .01), and nitrofurantoin (aOR = 1.57, 95% CI = 1.13-2.17, P = .01). CONCLUSIONS: Rural community type may influence urinary E. coli resistance to fluoroquinolones, SXT, and nitrofurantoin, indicating the need for antimicrobial stewardship interventions in medically underserved populations.

Genomic Insights into Drug Resistance Determinants in Cedecea neteri, A Rare Opportunistic Pathogen.

Cedecea, a genus in the Enterobacteriaceae family, includes several opportunistic pathogens reported to cause an array of sporadic acute infections, most notably of the lung and bloodstream. One species, Cedecea neteri, is associated with cases of bacteremia in immunocompromised hosts and has documented resistance to different antibiotics, including ß-lactams and colistin. Despite the potential to inflict serious infections, knowledge about drug resistance determinants in Cedecea is limited. In this study, we utilized whole-genome sequence data available for three environmental strains (SSMD04, M006, ND14a) of C. neteri and various bioinformatics tools to analyze drug resistance genes in this bacterium. All three genomes harbor multiple chromosome-encoded ß-lactamase genes. A deeper analysis of ß-lactamase genes in SSMD04 revealed four metallo-ß-lactamases, a novel variant, and a CMY/ACT-type AmpC putatively regulated by a divergently transcribed AmpR. Homologs of known resistance-nodulation-cell division (RND)-type multidrug efflux pumps such as OqxB, AcrB, AcrD, and MdtBC were also identified. Genomic island prediction for SSMD04 indicated that tolC, involved in drug and toxin export across the outer membrane of Gram-negative bacteria, was acquired by a transposase-mediated genetic transfer mechanism. Our study provides new insights into drug resistance mechanisms of an environmental microorganism capable of behaving as a clinically relevant opportunistic pathogen.

Clinical Management of COVID-19: A Review of Pharmacological Treatment Options.

Since the outbreak and subsequent declaration of COVID-19 as a global pandemic in March 2020, concerted efforts have been applied by the scientific community to curtail the spread of the disease and find a cure. While vaccines constitute a vital part of the public health strategy to reduce the burden of COVID-19, the management of this disease will continue to rely heavily on pharmacotherapy. This study aims to provide an updated review of pharmacological agents that have been developed and/or repurposed for the treatment of COVID-19. To this end, a comprehensive literature search was conducted using the PubMed, Google Scholar, and LitCovid databases. Relevant clinical studies on drugs used in the management of COVID-19 were identified and evaluated in terms of evidence of efficacy and safety. To date, the FDA has approved three therapies for the treatment of COVID-19 Emergency Use Authorization: convalescent plasma, remdesivir, and casirivimab/imdevimab (REGN-COV2). Drugs such as lopinavir/ritonavir, umifenovir, favipiravir, anakinra, chloroquine, hydroxychloroquine, tocilizumab, interferons, tissue plasminogen activator, intravenous immunoglobulins, and nafamosat have been used off-label with mixed therapeutic results. Adjunctive administration of corticosteroids is also very common. The clinical experience with these approved and repurposed drugs is limited, and data on efficacy for the new indication are not strong. Overall, the response of the global scientific community to the COVID-19 pandemic has been impressive, as evident from the volume of scientific literature elucidating the molecular biology and pathophysiology of SARS-CoV-2 and the approval of three new drugs for clinical management. Reviewed studies have shown mixed data on efficacy and safety of the currently utilized drugs. The lack of standard treatment for COVID-19 has made it difficult to interpret results from most of the published studies due to the risk of attribution error. The long-term effects of drugs can only be assessed after several years of clinical experience; therefore, the efficacy and safety of current COVID-19 therapeutics should continue to be rigorously monitored as part of post-marketing studies.

Sexually Transmitted Neisseria gonorrhoeae Infections-Update on Drug Treatment and Vaccine Development.

Background: Sexually transmitted gonorrhea, caused by the Gram-negative diplococcus Neisseria gonorrhoeae, continues to be a serious global health challenge despite efforts to eradicate it. Multidrug resistance among clinical N. gonorrhoeae isolates has limited treatment options, and attempts to develop vaccines have not been successful. Methods: A search of published literature was conducted, and information extracted to provide an update on the status of therapeutics and vaccine development for gonorrheal infection. Results: Recommended pharmacological treatment for gonorrhea has changed multiple times due to increasing acquisition of resistance to existing antibiotics by N. gonorrhoeae. Only broad-spectrum cephalosporin-based combination therapies are currently recommended for treatment of uncomplicated urogenital and anorectal gonococcal infections. With the reported emergence of ceftriaxone resistance, successful strategies addressing the global burden of gonorrhea must include vaccination. Century-old efforts at developing an effective vaccine against gonorrhea, leading to only four clinical trials, have not yielded any successful vaccine. Conclusions: While it is important to continue to explore new drugs for the treatment of gonorrhea, the historical trend of resistance acquisition suggests that any long-term strategy should include vaccine development. Advanced technologies in proteomics and in silico approaches to vaccine target identification may provide templates for future success.

Assessment and clinical utility of pharmacogenomics by healthcare practitioners in North Carolina.

Aim: Perceived knowledge, use and perceptions of pharmacogenomics (PGx) testing were assessed among healthcare practitioners in North Carolina. Materials & methods: A validated survey was distributed to various healthcare professionals and analyzed for differences among the groups. Results: The majority of the 744 survey respondents acknowledged the perceived benefits of PGx testing, but indicated either never or rarely using it. A substantial percentage of practitioners reported educational experiences but the majority had received no training. Among groups reporting using PGx testing, barriers to implementation were cost and insufficient training. Conclusion: The perceived cost of PGx testing and insufficiency or lack of training are major contributing factors to the infrequent use of PGx testing by healthcare providers in North Carolina.

Expanding spectrum of opportunistic Cedecea infections: Current clinical status and multidrug resistance.

Members of the bacterial genus Cedecea cause acute infections worldwide in compromised hosts with serious underlying medical conditions. While global reports of Cedecea infections remain sporadic in the medical literature, cases of multidrug-resistant clinical isolates have been documented each year over the past decade, warranting a comprehensive update on this emerging opportunistic pathogen. Here, we review the clinical manifestations, pathogenesis, natural distribution, epidemiology, and antimicrobial resistance of Cedecea species. Acute infection commonly manifests as bacteremia and pneumonia; however, the spectrum of infectious pathologies associated with Cedecea has expanded to include oral and cutaneous ulcers, orbital cellulitis, and peritonitis. The frequency of resistance among reported clinical isolates was highest to ampicillin, cephalothin, cefoxitin, cefazolin, and ceftazidime. Cedecea isolates harboring metallo-ß-lactamases exhibited resistance to carbapenems and fourth-generation cephalosporins. Further research is needed to understand the pathogenicity and multidrug resistance of Cedecea species. Appropriate therapeutic management of Cedecea infections depends on antibiotic susceptibility testing because of variable resistance patterns and the enhanced infection risk in vulnerable populations.

Indwelling medical device use and sepsis risk at a health professional shortage area hospital: Possible interaction with length of hospitalization.

BACKGROUND: We aimed to identify risk factors for sepsis diagnosis and possible interaction with length of hospital stay (LOS) among inpatients at a rural Health Professional Shortage Area hospital. METHODS: This case-control study examined 600 adult patients (300 cases and 300 controls) admitted to a rural health system in North Carolina between 2012 and 2018. Case selection was based on assignment of ICD-9-CM diagnostic codes for sepsis. Controls were patients with a medical diagnosis other than sepsis during the observational period. Logistic regression was used to model sepsis diagnosis as a function of indwelling medical device use and stratified by LOS. RESULTS: Indwelling medical device use preadmission and postadmission were significantly associated with increased risk of sepsis diagnosis among patients with extended hospital stays (LOS ≥ 5 days) (odds ratio [OR] = 5.51; 95% confidence interval [CI] = 1.95-15.62; P = .001 and OR = 3.28; 95% CI = 1.24-8.68; P = .017, respectively). Among patients with LOS <5 days, association with sepsis diagnosis was only significant for indwelling medical device use preadmission (OR = 9.61; 95% CI = 3.68-25.08; P < .0001). CONCLUSIONS: Indwelling medical device use was significantly associated with increased risk of sepsis diagnosis and the risk was higher with longer hospitalization.

Urinary Catheter Colonization by Multidrug-Resistant Cedecea neteri in Patient with Benign Prostatic Hyperplasia.

Cedecea neteri, a member of the Enterobacteriaceae family, has only been identified as a human pathogen in a few previous clinical cases, thus complicating assessment of this organism's pathogenicity and medical relevance. Documented infections attributed to C. neteri primarily involved bacteremia in severely immunocompromised patients. We report a rare case of urinary catheter colonization by a multidrug-resistant C. neteri strain in a patient of advanced age with benign prostatic hyperplasia and other chronic comorbidities. This C. neteri isolate was resistant or intermediate to second-generation cephalosporins, penicillins, and certain ß-lactamase inhibitor/ß-lactam combinations. Analysis of whole genome sequence information for a representative C. neteri strain indicated the presence of multiple open reading frames with sequence similarity to ß-lactamases, including a chromosome-encoded AmpC ß-lactamase and metallo-ß-lactamases, consistent with the resistance phenotype of this bacterium. The presence of an AmpR homolog suggests that the C. neteriampC may be inducible in response to ß-lactam exposure. Molecular insights into antibiotic resistance traits of this emerging opportunistic pathogen will be important for administering adequate antibiotic treatment to ensure favorable patient outcomes.

Risk factors for sepsis morbidity in a rural hospital population: A case-control study.

BACKGROUND: The aim of the study was to identify risk factors for sepsis morbidity in a rural hospital population. METHODS: We used a case-control study design. Patients included adult admissions to a rural health system between January 1, 2012, and December 31, 2015. Case selection was by electronic medical record search for codes of the ICD-9-CM. Cases were validated against Quick Sequential Organ Failure Assessment criteria. Multiple logistic regression modeling was performed to determine which predefined variables were significantly associated with sepsis diagnosis. RESULTS: A total of 220 patients were studied (110 cases and 110 controls). Cases had an in-hospital mortality of 20% compared with 0% of the controls. Indwelling medical device use during hospitalization (adjusted odds ratio [OR], 3.02; 95% confidence interval [CI], 1.44-6.30; P = .003), coronary heart disease (adjusted OR, 2.59; 95% CI, 1.13-5.97; P = .03), and type of health insurance (adjusted OR, 2.36; 95% CI, 1.13-4.93; P = .02) were independently associated with sepsis diagnosis after adjusting for potential confounders. CONCLUSIONS: This study underscores the need for implementation and maintenance of infection control measures during management of patients with indwelling medical devices at a rural hospital.

Hepatitis C Virus-Genotype 3: Update on Current and Emergent Therapeutic Interventions.

PURPOSE OF REVIEW: Direct-acting antiviral agents (DAAs) have markedly improved the prognosis of hepatitis C virus (HCV)-genotype 3 (GT3), a highly prevalent infection worldwide. However, in patients with hepatic fibrosis, cirrhosis, or hepatocellular carcinoma (HCC), GT3 infection presents a treatment challenge compared with other genotypes. The dependence of the HCV life cycle on host lipid metabolism suggests the possible utility of targeting host cellular factors for combination anti-HCV therapy. We discuss current and emergent DAA regimens for HCV-GT3 treatment. We then summarize recent research findings on the reliance of HCV entry, replication, and virion assembly on host lipid metabolism. RECENT FINDINGS: Current HCV treatment guidelines recommend the use of daclatasvir plus sofosbuvir (DCV/SOF) or sofosbuvir plus velpatasvir (SOF/VEL) for the management of GT3 based upon clinical efficacy [≥88% overall sustained virological response (SVR)] and tolerability. Potential future DAA options, such as SOF/VEL co-formulated with GS-9857, also look promising in treating cirrhotic GT3 patients. However, HCV resistance to DAAs will likely continue to impact the therapeutic efficacy of interferon-free treatment regimens. Disruption of HCV entry by targeting required host cellular receptors shows potential in minimizing HCV resistance and broadening therapeutic options for certain subpopulations of GT3 patients. The use of cholesterol biosynthesis and transport inhibitors may also improve health outcomes for GT3 patients when used synergistically with DAAs. Due to the morbidity and mortality associated with HCV-GT3 infection compared to other genotypes, efforts should be made to address current limitations in the therapeutic prevention and management of HCV-GT3 infection.

Intracellularly grown gold nanoislands as SERS substrates for monitoring chromate, sulfate and nitrate localization sites in remediating bacteria biofilms by Raman chemical imaging.

Understanding the chemical composition of biofilm matrices is vital in different fields of biology such as surgery, dental medicine, synthetic grafts and bioremediation. The knowledge of biofilm development, composition, active reduction sites and remediation efficacy will help in the development of effective solutions and evaluation of remediating approaches prior to implementation. Surface-enhanced Raman spectroscopy (SERS) based imaging is an invaluable tool to obtain an understanding of the remediating efficacy of microorganisms and its role in the formation of organic and inorganic compounds in biofilms. We demonstrate for the first time, the presence of chromate, sulfate, nitrate and reduced trivalent chromium in soil biofilms. In addition, we demonstrate that SERS imaging was able to validate two observations made by previous studies on chromate/sulfate and chromate/nitrate interactions in Shewanella oneidensis MR-1 biofilms. Additionally, we show a detailed Raman mapping based evidence of the existence of chromate-sulfate competition for cellular entry. Subsequently, we use Raman mapping to study the effect of nitrate on chromate reduction. The findings presented in this paper are among the first to report - detection of multiple metallic ions in bacterial biofilms using intracellular SERS substrates. Such a detailed characterization of biofilms using gold nanoislands based SERS mapping substrate can be extended to study cellular localization of other metallic ions and chemical species of biological and toxicological significance and their effect on reduction reactions in bacterial biofilms.

Raman chemical imaging of chromate reduction sites in a single bacterium using intracellularly grown gold nanoislands.

Imaging live molecular events within micro-organisms at single-cell resolution would deliver valuable mechanistic information much needed in understanding key biological processes. We present a surface-enhanced Raman (SERS) chemical imaging strategy as a first step toward exploring the intracellular bioreduction pockets of toxic chromate in Shewanella. In order to achieve this, we take advantage of an innate reductive mechanism in bacteria of reducing gold ions into intracellular gold nanoislands, which provide the necessary enhancement for SERS imaging. We show that SERS has the sensitivity and selectivity not only to identify but also to differentiate between the two stable valence forms of chromate in cells. The imaging platform was used to understand intracellular metal reduction activities in a ubiquitous metal-reducing organism, Shewanella oneidensis MR-1, by mapping chromate reduction.

SO2426 is a positive regulator of siderophore expression in Shewanella oneidensis MR-1.

BACKGROUND: The Shewanella oneidensis MR-1 genome encodes a predicted orphan DNA-binding response regulator, SO2426. Previous studies with a SO2426-deficient MR-1 strain suggested a putative functional role for SO2426 in the regulation of iron acquisition genes, in particular, the siderophore (hydroxamate) biosynthesis operon so3030-3031-3032. To further investigate the functional role of SO2426 in iron homeostasis, we employed computational strategies to identify putative gene targets of SO2426 regulation and biochemical approaches to validate the participation of SO2426 in the control of siderophore biosynthesis in S. oneidensis MR-1. RESULTS: In silico prediction analyses revealed a single 14-bp consensus motif consisting of two tandem conserved pentamers (5'-CAAAA-3') in the upstream regulatory regions of 46 genes, which were shown previously to be significantly down-regulated in a so2426 deletion mutant. These genes included so3030 and so3032, members of an annotated siderophore biosynthetic operon in MR-1. Electrophoretic mobility shift assays demonstrated that the SO2426 protein binds to its motif in the operator region of so3030. A "short" form of SO2426, beginning with a methionine at position 11 (M11) of the originally annotated coding sequence for SO2426, was also functional in binding to its consensus motif, confirming previous 5' RACE results that suggested that amino acid M11 is the actual translation start codon for SO2426. Alignment of SO2426 orthologs from all sequenced Shewanella spp. showed a high degree of sequence conservation beginning at M11, in addition to conservation of a putative aspartyl phosphorylation residue and the helix-turn-helix (HTH) DNA-binding domain. Finally, the so2426 deletion mutant was unable to synthesize siderophores at wild-type rates upon exposure to the iron chelator 2,2'-dipyridyl. CONCLUSIONS: Collectively, these data support the functional characterization of SO2426 as a positive regulator of siderophore-mediated iron acquisition and provide the first insight into a coordinate program of multiple regulatory schemes controlling iron homeostasis in S. oneidensis MR-1.

Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

Proteomics reveals a core molecular response of Pseudomonas putida F1 to acute chromate challenge.

BACKGROUND: Pseudomonas putida is a model organism for bioremediation because of its remarkable metabolic versatility, extensive biodegradative functions, and ubiquity in contaminated soil environments. To further the understanding of molecular pathways responding to the heavy metal chromium(VI) [Cr(VI)], the proteome of aerobically grown, Cr(VI)-stressed P. putida strain F1 was characterized within the context of two disparate nutritional environments: rich (LB) media and minimal (M9L) media containing lactate as the sole carbon source. RESULTS: Growth studies demonstrated that F1 sensitivity to Cr(VI) was impacted substantially by nutrient conditions, with a carbon-source-dependent hierarchy (lactate > glucose >> acetate) observed in minimal media. Two-dimensional HPLC-MS/MS was employed to identify differential proteome profiles generated in response to 1 mM chromate under LB and M9L growth conditions. The immediate response to Cr(VI) in LB-grown cells was up-regulation of proteins involved in inorganic ion transport, secondary metabolite biosynthesis and catabolism, and amino acid metabolism. By contrast, the chromate-responsive proteome derived under defined minimal growth conditions was characterized predominantly by up-regulated proteins related to cell envelope biogenesis, inorganic ion transport, and motility. TonB-dependent siderophore receptors involved in ferric iron acquisition and amino acid adenylation domains characterized up-regulated systems under LB-Cr(VI) conditions, while DNA repair proteins and systems scavenging sulfur from alternative sources (e.g., aliphatic sulfonates) tended to predominate the up-regulated proteome profile obtained under M9L-Cr(VI) conditions. CONCLUSIONS: Comparative analysis indicated that the core molecular response to chromate, irrespective of the nutritional conditions tested, comprised seven up-regulated proteins belonging to six different functional categories including transcription, inorganic ion transport/metabolism, and amino acid transport/metabolism. These proteins might potentially serve as indicators of chromate stress in natural microbial communities.

High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes.

BACKGROUND: The genome of Arthrobacter sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes chrA, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge. RESULTS: A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of chrA expression were achieved when the chrB orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles. CONCLUSION: Our findings indicate that chromate resistance in Arthrobacter sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study.

Global proteomic analysis of the chromate response in Arthrobacter sp. strain FB24.

A global proteomic evaluation of the response of Arthrobacter sp. strain FB24 to 5 and 20 mM Cr(VI) was conducted using both two-dimensional gel electrophoresis (2-DGE) and liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS). The changes in protein expression found with 2-DGE indicate alterations in central metabolism and amino acid synthesis. Proteome coverage increased from 22% with 2-DGE to 71% with LC/LC-MS/MS. The proteins exhibiting the highest levels of expression under Cr(VI) stress suggest intracellular sulfur limitation, which could be driven by competition for the sulfate (SO4(2-)) transporter by the chromate (CrO4(2-)) ion. These results are consistent with the growth defects seen with strain FB24 when Cr(VI) concentrations exceeded 5 mM.