Pseudomonas aeruginosa deficient in 8-oxodeoxyguanine repair system shows a high frequency of resistance to ciprofloxacin

The 8-oxodeoxyguanine (8-oxodG) repair system participates in the prevention and correction of mutations generated by oxidative DNA damage in prokaryotes and eukaryotes. In this study, we report that Pseudomonas aeruginosa strains deficient in this repair mechanism by inactivation of the mutT, mutM and mutY genes generate a high frequency of cells resistant to the antibiotic ciprofloxacin. In the mutT strain, the increase in ciprofloxacin resistance achieved at threefold minimal inhibitory concentration was about 1600-fold over the wild-type (WT) level, similar to the frequency achieved by the mismatch repair-deficient mutS strain. Molecular analysis of WT, mutT and mutY clones resistant to ciprofloxacin indicated that the nfxB gene was mutated in the majority of the cases, while mutS- derived resistant clones were mainly mutated in gyrA and parC genes. Cell viability analysis after treatment with paraquat or hydrogen peroxide indicated that 8-oxodG repair-deficient strains were considerably more susceptible to oxidative stress than the parental strain. Finally, it is shown that the ciprofloxacin resistance frequency of WT and repair-deficient strains increased significantly after cell exposure to paraquat. Thus, oxidative stress is strongly implicated in the emergence of ciprofloxacin-resistant mutants in P. aeruginosa , and the 8-oxodG repair pathway plays an important role in the prevention of these mutations.     

奇异变形杆菌耐药性及亚胺培南不敏感株感染的临床特点研究

目的研究奇异变形杆菌的临床分布和耐药情况、亚胺培南不敏感奇异变形杆菌感染的临床特点。方法分析浙江大学医学院附属第一医院2013年1月至2013年12月分离的非重复奇异变形杆菌的药物敏感性、临床分布,回顾性分析亚胺培南不敏感奇异变形杆菌感染患者的临床资料、治疗及预后情况。结果2013年该院共分离107株奇异变形杆菌,以分离自尿液最多,其次为痰液;来源最多的是外科病房和重症监护病房。体外药敏显示：奇异变形杆菌对美罗培南、厄他培南、头孢吡肟、氨曲南、哌拉西林/他唑巴坦、头孢他啶、头孢哌酮/舒巴坦、阿米卡星等抗菌药物敏感性良好,敏感率达85%以上;对亚胺培南敏感率为80.4%;对头孢呋辛、环丙沙星、氨苄西林、头孢曲松、庆大霉素耐药率较高,超过30%;对呋喃妥因耐药率为99%。其中21株亚胺培南不敏感奇异变形杆菌对包括美罗培南、厄他培南在内的其他各类抗菌药物耐药率与亚胺培南敏感株基本相仿。亚胺培南不敏感奇异变形杆菌引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,可引起泌尿系统、皮肤创面、腹腔、血流、生殖道等部位感染,表现为全身炎症反应及局部感染症状。选择敏感抗菌药物治疗后该部分患者预后良好。结论奇异变形杆菌对三、四代头孢菌素,β-内酰胺酶抑制剂合剂等抗生素敏感性良好。亚胺培南不敏感奇异变形杆菌对其他碳青酶烯类抗生素仍保持较高的敏感性。亚胺培南不敏感奇异变形杆菌所引起院内获得性感染主要发生在入住ICU、外科术后、广谱抗菌药物使用后、留置各类置管和梗阻性尿路疾病的患者,预后良好。     

Increased resistance to first-line agents among bacterial pathogens isolated from urinary tract infections in Latin America: time for local guidelines?

Emerging resistance phenotypes and antimicrobial resistance rates among pathogens recovered from community-acquired urinary tract infections (CA-UTI) is an increasing problem in specific regions, limiting therapeutic options. As part of the SENTRY Antimicrobial Surveillance Program, a total of 611 isolates were collected in 2003 from patients with CA-UTI presenting at Latin American medical centers. Each strain was tested in a central laboratory using Clinical Laboratory Standard Institute (CLSI) broth microdilution methods with appropriate controls. Escherichia coli was the leading pathogen (66%), followed by Klebsiella spp. (7%), Proteus mirabilis (6.4%), Enterococcus spp. (5.6%), and Pseudomonas aeruginosa (4.6%). Surprisingly high resistance rates were recorded for E. coli against first-line orally administered agents for CA-UTI, such as ampicillin (53.6%), TMP/SMX (40.4%), ciprofloxacin (21.6%), and gatifloxacin (17.1%). Decreased susceptibility rates to TMP/SMX and ciprofloxacin were also documented for Klebsiella spp. (79.1 and 81.4%, respectively), and P. mirabilis (71.8 and 84.6%, respectively). For Enterococcus spp., susceptibility rates to ampicillin, chloramphenicol, ciprofloxacin, and vancomycin were 88.2, 85.3, 55.9, and 97.1%, respectively. High-level resistance to gentamicin was detected in 24% of Enterococcus spp. Bacteria isolated from patients with CA-UTI in Latin America showed limited susceptibility to orally administered antimicrobials, especially for TMP/SMX and fluoroquinolones. Our results highlight the need for developing specific CA-UTI guidelines in geographic regions where elevated resistance to new and old compounds may influence prescribing decisions.     

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In Gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl β-naphtylamide (PAβN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256–0.25 μg/ml) with a fixed concentration of PAβN (50 μg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAβN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAβN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.     

Influence of recA mutations on gyrA dependent quinolone resistance.

We examined, in Escherichia coli, the influence of recA mutant alleles on the level of quinolone resistance promoted by mutations in the gyrA gene. We found that the recA142 mutation, abolishing all the activities of RecA protein, greatly reduced the level of resistance to the quinolone ciprofloxacin, whereas the recA430 allele affecting the SOS inducing ability of RecA, reduced ciprofloxacin resistance to a lesser extent. The recA142 mutation did not cause enhancement of ciprofloxacin induced DNA breakage in gyrA mutants, indicating that the stabilization of DNA-gyrase complexes by the quinolone is not influenced by a RecA mutant protein. We suggest that RecA protein plays a role in the repair of quinolone damage, principally through a recombinational mechanism and, to a lesser degree, through the induction of the SOS response.     

Development of fluoroquinolone (ciprofloxacin) resistance in Mycoplasma hominis in the presence of Hela cells

The effect of cocultivation of eukaryotic HeLa cells and Mycoplasma hominis mycoplasma on the resistance of the latter to fluoroquinolones (ciprofloxacin) was examined. It was shown that cocultivation of the M. homonis and HeLa cells during 24 h with subsequent addition of ciprofloxacin resulted in an increase of the mircoplasma resistance to this antimicrobial agent. In the M. hominis cells cultivated in the presence of HeLa cells and the increasing concentration of ciprofloxacin mutations in the parC gene were observed only at low concentrations of the antimicrobial agent, while mutations in the gyrA gene were never detected. A gradual elevation of ciprofloxacin concentration up to 10 micrograms/ml resulted in the reversion of the parC mutations in mycoplasmas. Mycoplasma cells resistant to high flouroquinolone concentrations and isolated after cocultivation with the HeLa cells were characterized by the wild-type genotype in respect of the gyrA and parC genes. It was shown for the first time that infection of HeLa cells resulted in the appearance of genome rearrangements in M. hominis cells.     

Mutation in mexR-gene leading to drug resistance in corneal keratitis in human

The present study deals with the genetic polymorphism of the mexR gene which is involved in the resistance to drugs like ciprofloxacin. Mutations in mexR result in increased resistance to multiple antibiotics due to overexpression of this efflux system. The MexR product contains 147 amino acids with a molecular mass of 16,964 Da. We detected 28 point mutations in 14 samples from corneal scraping infected with Pseudomonas aeruginosa, which were screened for ciprofloxacin resistance. Twenty four were silent mutations and four missense mutations. Mapping these mutations was done by using in silico methods on the protein 3D- structure obtained from PDB database, localized at 3 specific sites. Single amino acid changes (mutations) may influence MexR stability or its ability to dimerise, and thus result in the conformation changes at the DNA-binding domain, of the structure. Hence it is concluded that the mutations in the DNA binding domain of mexR gene could be one of the factors contributing to the possible drug resistance in these patients.     

Ciprofloxacin induces mutagenesis to antibiotic resistance independent of UmuC in Streptococcus uberis

Streptococcus uberis is an environmental bovine mastitis pathogen capable of UV-inducible SOS mutagenesis. Bacterial SOS systems can be induced by several chemicals including also antibiotics used in clinical practice. Here, we have studied the effect of ciprofloxacin, a fluoroquinolone antibiotic and known inducer of SOS, on mutations leading to antibiotic resistance in S. uberis . Mutation frequencies and spectra were compared in a wild-type S. uberis strain and its Δ umuC derivative. The results revealed that concentrations of ciprofloxacin corresponding to 0.3–0.5× minimum inhibitory concentration (MIC) induce mutagenesis independent of UmuC. Partial sequencing of the rpoB gene of individual rifampin-resistant clones from wild-type and Δ umuC strains revealed a similar but complex pattern of point mutations including transitions, transversions and deletions/insertions. It was previously shown that UV induces mainly transition-type mutations and UmuC is essential for the process. Thus, the results presented here demonstrate that S. uberis employs distinct mechanisms for ciprofloxacin and UV-induced mutagenesis, which is a striking difference to Escherichia coli SOS model.     

Fungal cytochrome P450 protein Cyp51: What we can learn from its evolution,regulons and Cyp51-based azole resistance

Cyp51 (Sterol 14α-demethylase) is the single cytochrome P450 (Cyp) required for sterol biosynthesis in different phyla. Among hundreds of P450 proteins, Cyp51 is evolutionarily the oldest P450 protein and is the only cytochrome P450 protein present in most biological kingdoms including fungi, bacteria, plants and animals. A valuable class of antifungals such as azoles, amphotericin B, specifically target the fungal Cyp51 (Erg11), a lanosterol demethylase that is critical for the specific component of the fungal plasma membrane ergosterol biosynthesis. However, pathogenic fungi worldwide have developed resistance to azoles, largely through mutations in the Cyp51/Erg11 protein. Structural studies have elucidated the resistance mechanisms associated with these mutations are mostly caused by decreased the binding affinity of the azoles to the Cyp51 protein and affect the stability of Cyp51 protein. In addition, the overexpression of the cyp51 gene will also increase azole resistance, which addresses the critical role of Cyp51 regulators. In this review, we explore the fungal Cyp51 from the evolution, regulation and the contribution of Cyp51 mutations to azole resistance aspects. Knowledge gained from Cyp51 research will benefit to develop novel Cyp51-based antifungals.     

Proteomic analysis of Proteus mirabilis outer membrane proteins reveals differential expression in vivo vs. in vitro conditions

Proteus mirabilis is an opportunistic pathogen that frequently causes complicated urinary tract infections. Among a wide spectrum of potential virulence factors, outer membrane proteins (OMPs) are critical for bacterial interactions and survival in different environments. In this work, we used a proteomic approach to assess P. mirabilis in vivo OMPs expression compared to in vitro, including iron replete and iron-restricted conditions. Three putative iron receptors, IreA, PMI0842, and PMI2596, were detected both in bacterium grown in vivo and in vitro under iron-restricted conditions. A prophage gene product, PMI1721, was detected only on in vivo growing bacterium, suggesting a potential role yet to be disclosed on the surface of P. mirabilis. Plasminogen, a host protein, was co-purified with OMPs of in vivo grown bacteria, which is in accordance with previous observations and suggests that plasminogen bound to P. mirabilis surface may be associated to virulence as seen in other bacterial pathogens. Western blots using sera of experimentally challenged mice showed that iron-regulated proteins are expressed and highly immunogenic during infection. This work confirms observations made by others for P. mirabilis and reveals details not yet described, suggesting new aspects of the bacterium pathogenesis that remain unknown.     

Covalently linked kanamycin – Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance

To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The hybrids were antagonistic relative to the ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides. Selected hybrids were 42–640 fold poorer inhibitors of bacterial protein synthesis than the parent kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance development in both E. coli and B. subtilis in comparison to that of component drugs alone or their 1:1 mixture. More generally, the data suggest that an antagonistic combination of aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the emergence of resistance.     

Effects of black tea consumption on plasma catechins and markers of oxidative stress and inflammation in patients with coronary artery disease

We previously demonstrated that black tea consumption reverses endothelial dysfunction in patients with coronary artery disease. To investigate potential mechanisms of this effect, we examined plasma catechins and systemic markers of oxidation, inflammation, and antioxidant protection from 66 subjects enrolled in that study. We collected samples at baseline, 2 h after 450 ml of black tea (acute), after 4 weeks of 900 ml of black tea per day (chronic), and after acute and chronic consumption of water. Total catechins increased 33% after acute tea (P < 0.05) and 29% after chronic tea (P < 0.05). Of individual catechins, plasma epicatechin gallate (ECG) concentration significantly increased with acute tea consumption, and plasma epicatechin (EC) increased with chronic tea consumption. Tea consumption did not improve plasma antioxidant capacity and did not reduce urinary 8-hydroxy-2'-deoxyguanosine, or urinary 8-isoprostane levels. Changes in catechin levels did not correlate with changes in endothelial function, plasma markers of oxidative stress, or C-reactive protein. In contrast, endothelial function at baseline correlated with dietary flavonoid intake (beta = 0.32, P = 0.02) and with baseline plasma EC concentration after adjusting for confounding variables (beta = 0.39, P = 0.03). These findings suggest that the benefits of black tea consumption on endothelial function may not be attributable to tea catechins or a systemic antioxidant or anti-inflammatory effect. Chronic dietary flavonoid status appears to relate to endothelial function, possibly suggesting that other flavonoids or polyphenolic components of tea favorably influence vascular health and risk for cardiovascular disease.     

Macromolecular Oxidation in Planktonic Population and Biofilms of Proteus mirabilis Exposed to Ciprofloxacin

Diverse chemical and physical agents can alter cellular functions associated with the oxidative metabolism, thus stimulating the production of reactive oxygen species (ROS). Proteins and lipids may be important targets of oxidation, and this may alter their functions in planktonic bacterial physiology. However, more research is necessary to determine the precise role of cellular stress and macromolecular oxidation in biofilms. The present study was designed to evaluate whether ciprofloxacin (CIP) could oxidize the lipids to malondialdehyde (MDA) and the proteins to carbonyl residues and to advanced oxidation protein products (AOPP) in planktonic populations and biofilms of Proteus mirabilis. Incubation with CIP generated an increase of lipid and protein oxidation in planktonic cells, with a greater effect found in sensitive strains than resistant ones. Biofilms showed higher basal levels of oxidized macromolecules than planktonic bacteria, but there was no significant enhancement of MDA, carbonyl, or AOPP with antibiotic. The results described in this article show the high basal levels of MDA, carbonyls, and AOPP, with aging and loss of proliferation of biofilms cells. The low response to the oxidative stress generated by CIP in biofilms helps to clarify the resistance to antibiotics of P. mirabilis when adhered to surfaces.     

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.     

奇异变形杆菌中基因岛介导的多重耐药传播研究进展

奇异变形杆菌是导致医院内感染的重要条件致病菌,广泛分布于自然环境及人和动物的肠道中。基因岛是细菌染色体上约10-200 kb独立的DNA片段,能促进宿主细菌适应复杂多变的环境,与细菌适应性进化密切相关。近年来在奇异变形杆菌基因组中发现了多个与多重耐药密切相关的基因岛,包括沙门菌基因岛1及其相关基因岛、SXT/R391整合性接合元件、PmGRI1等,表明基因岛在奇异变形杆菌多重耐药形成和传播中具有重要作用。本文对奇异变形杆菌中与耐药相关基因岛的结构特征、传播机制、流行情况等进行综述,以期为奇异变形杆菌中多重耐药相关基因岛的深入研究提供参考。     

一株奇异变形杆菌对铜绿假单胞菌多细胞行为的抑制作用

目的：抗生素耐药性成为了全球性的健康问题。研究发现病原菌的多细胞行为在抗生素的耐药性中起着至关重要的作用（尤其是生物膜）,因而通过抑制多细胞行为而控制耐药性成为当务之急。本文以奇异变形杆菌（Proteus mirabilis）为研究对象,考察它的发酵滤液对一种机会致病菌——铜绿假单胞菌（Pseudomonas aeruginosa）多细胞行为的作用,以期得到一株多细胞行为抑制菌：在不影响Paerugiliosa生长的前提下,抑制生物膜形成、EPS产生以及定向丛集运动,解除保护,减缓扩散,为降低Paemgi—nosa耐药性,增强抗生素作用效果提供可能。方法：采用结晶紫生物膜测定法、蒽酮一硫酸法、平板检测法,探究Pmirabilis发酵滤液对Paemginosa生物膜、胞外多聚物、定向丛集运动和生长的影响。结果：Pmirabilis发酵滤液能显著抑制Paeruginosa生物膜量,在体积百分比浓度为1％时,抑制率可达60．9％。该菌的发酵滤液还能阻碍Paeruginosa的定向丛集运动,减弱它的吸附和扩散运动;同时,也减少了Pacrugillosa胞外多聚物的产量,在滤液体积百分比浓度为1％时,抑制率达到45．9％。更重要的是,固体平板实验证明该发酵滤液对P．aemginosa的生长没有影响。结论：Pmirabilis在不影响病原菌生长的前提下,对病原菌的多细胞行为有一定的控制作用。其发酵滤液中存在着抑制微生物膜、定向丛集运动等的成分,在治疗细菌感染性疾病和降低抗生素耐药性方面有潜在应用价值。     

Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections

Proteus mirabilis is a common cause of urinary tract infection (UTI) and produce several types of different fimbriae, including mannose-resistant/Proteus-like fimbriae, uroepithelial cell adhesin (UCA), and P. mirabilis fimbriae (PMF). Different authors have related these fimbriae with different aspects of P. mirabilis pathogenesis, although the precise role of fimbriae in UTI has not yet been elucidated. In this work we expressed and purified recombinant structural fimbrial proteins of these fimbriae (MrpA, UcaA, and PmfA) and assessed their role as protective antigens using an ascending and a haematogenous model of UTI in the mouse. MrpA protected subcutaneously immunised mice in both models, suggesting that it could be taken into account as a promising vaccine candidate against P. mirabilis UTI. UcaA could also be an interesting subunit to be studied although it only protected mice that were challenged intravenously. All subunits elicited a strong specific serum IgG response but there was no significant correlation between antibody levels and protection. Only PmfA-immunised mice elicited a significant urinary antibody response but this protein was unable to confer protection against P. mirabilis experimental challenges. These results may contribute to the development of vaccines against P. mirabilis, an important cause of complicated UTI.     

Changes in Acholeplasma laidlawii plasma membrane functions during development of resistance to ciprofloxacin and tetracycline]

The role of transport activity of Acholeplasma laidlawii plasmatic membrane in the development of resistance to ciprofloxacin and tetracycline was investigated. It was shown that one of the important steps of resistance development in acholeplasms is a complex of adaptation metabolic reactions providing limited antibiotic accumulation by the cells. In the case of ciprofloxacin resistance metabolism changes concerning transport systems took place before mutations in target genes. Development of tetracycline resistance of mollecutes after incubation in the medium with enhancing antibiotic concentrations and not connected with the presence of tet(M) determinant was demonstrated for the first time.     

An important role of a "probable ATP-binding component of ABC transporter" during the process of Pseudomonas aeruginosa resistance to fluoroquinolone

In order to find new drug target to eliminate the fluoroquinolone resistance, the in vitro progress of Pseudomonas aeruginosa fluoroquinolone resistance was mimicked, and then proteomic analysis was applied to comparing different protein profiles during the resistant process. The results show that the expression of a "probable ATP-binding component of ATP binding cassette (ABC) transporter" existed in ciprofloxacin-intermediate and -resistant strains, but not in sensitive strain. In addition, the ciprofloxacin concentrations in P. aeruginosa strains, which were obtained from the progress of P. aeruginosa fluoroquinolone resistance, were determined by means of HPLC; the results show that the decrease of the intracellular concentration of drug and the expression of this new protein nearly take place simultaneously. The changes of mRNA levels of the probable ATP-binding component of ABC transporter were detected by virtue of RT-PCR and showed that this protein did not express in the sensitive strains but expressed increasingly in the intermediate and resistant strains. In order to determine the relationships between the development of antibiotic resistance and this protein further, a DNAzyme was designed to aim at the mRNA of the probable ATP-binding component of ABC transporter directly; the ciprofloxacin resistance of P. aeruginosa was partially reduced in vivo by inhibiting the expression of this protein. This DNAzyme has no effect on sensitive strain. And the comparison of drug intracellular concentrations between DNAzyme-treated strains and its control strains shows that this protein may be included in the course of active drug efflux.     

Fumarate reduction in Proteus mirabilis.

1. Proteus mirabilis formed fumarate reductase under anaerobic growth conditions. The formation of this reductase was repressed under conditions of growth during which electron transport to oxygen or to nitrate is possible. In two of three tested chlorate-resistant mutant strains of the wild type, fumarate reductase appeared to be affected. 2. Cytoplasmic membrane suspensions isolated from anaerobically grown P. mirabilis oxidized formate and NADH with oxygen and with fumarate, too. 3. Spectral investigation of the cytoplasmic membrane preparation revealed the presence of (probably at least two types of) cytochrome b, cytochrome a1 and cytochrome d. Cytochrome b was reduced by NADH as well as by formate to approximately 80%. 4. 2-n-Heptyl-4-hydroxyquinilone-N-oxide and antimycin A inhibited oxidation of both formate and NADH by oxygen and fumarate. Both inhibitors increased the level of the formate/oxygen steady state and the formate/fumarate steady state. 5. The site of inhibition of the respiratory activity by both HQNO and antimycin A was located at the oxidation side of cytochrome b. 6. The effect of ultraviolet-irradiation of cytoplasmic membrane suspensions on oxidation/reduction phenomena suggested that the role of menaquinone is more exclusive in the formate/fumarate pathway than in the electron transport route to oxygen. 7. Finally, the conclusion has been drawn that the preferential route for electron transport from formate and from NADH to fumarate (and to oxygen) includes cytochrome b as a directly involved carrier. A hypothetical scheme for the electron transport in anaerobically grown P. mirabilis is presented.