Microbiota and metabolite alterations in pancreatic head and body/tail cancer patients.

Pancreatic head cancer (PHC) and pancreatic body/tail cancer (PBTC) have distinct clinical and biological behaviors. The microbial and metabolic differences in PHC and PBTC have not been studied. The pancreatic microbiota and metabolome of 15 PHC and 8 PBTC tissues and their matched nontumor tissues were characterized using 16S rRNA amplicon sequencing and untargeted metabolomics. At the genus level, Bradyrhizobium was increased while Corynebacterium and Ruminococcus were decreased in the PHC tissues (Head T) compared with the matched nontumor tissues (Head N) significantly. Shuttleworthia, Bacillus, and Bifidobacterium were significantly decreased in the PBTC tissues (Body/Tail T) compared with the matched nontumor tissues (Body/Tail N). Significantly, Ileibacterium was increased whereas Pseudoxanthomonas was decreased in Head T and Body/Tail T, and Lactobacillus was increased in Head T but decreased in Body/Tail T. A total of 102 discriminative metabolites were identified between Head T and Head N, which were scattered through linoleic acid metabolism and purine metabolism pathways. However, there were only four discriminative metabolites between Body/Tail T and Body/Tail N, which were related to glycerophospholipid metabolism and autophagy pathways. The differential metabolites in PHC and PBTC were commonly enriched in alpha-linolenic acid metabolism and choline metabolism in cancer pathways. Eubacterium decreased in Head T was positively correlated with decreased linoleic acid while negatively correlated with increased arachidyl carnitine and stearoylcarnitine. Bacillus decreased in Body/Tail T was negatively correlated with increased L-carnitine. These microbiota and metabolites deserve further investigations to reveal their roles in the pathogenesis of PHC and PBTC, providing clues for future treatments.

Characterization of Chemical Interactions between Clinical Drugs and the Oral Bacterium, Corynebacterium matruchotii, via Bioactivity-HiTES.

In the field of natural product research, the rediscovery of already-known compounds is one of the significant issues hindering new drug development. Recently, an innovative approach called bioactivity-HiTES has been developed to overcome this limitation, and several new bioactive metabolites have been successfully characterized by this method. In this study, we applied bioactivity-HiTES to Corynebacterium matruchotii, the human oral bacterium, with 3120 clinical drugs as potential elicitors. As a result, we identified two cryptic metabolites, methylindole-3-acetate (MIAA) and indole-3-acetic acid (IAA), elicited by imidafenacin, a urinary antispasmodic drug approved by the Japanese Pharmaceuticals and Medical Devices Agency (PMDA). MIAA showed weak antibacterial activity against a pulmonary disease-causing Mycobacterium conceptionense with an IC50 value of 185.7 µM. Unexpectedly, we also found that C. matruchotii metabolized fludarabine phosphate, a USFDA-approved anticancer drug, to 2-fluoroadenine which displayed moderate antibacterial activity against both Bacillus subtilis and Escherichia coli, with IC50 values of 8.9 and 20.1 µM, respectively. Finally, acelarin, a prodrug of the anticancer drug gemcitabine, was found to exhibit unreported antibacterial activity against B. subtilis with an IC50 value of 33.6 µM through the bioactivity-HiTES method as well. These results indicate that bioactivity-HiTES can also be applied to discover biotransformed products in addition to finding cryptic metabolites in microbes.

Insights into the role of F26 residue in the FMN: ATP adenylyltransferase activity of Staphylococcus aureus FAD synthetase.

The bifunctional flavin adenine dinucleotide synthetase (FADS) synthesizes the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) co-factors essential for the function of flavoproteins. The Staphylococcus aureus FADS (SaFADS) produces FMN from riboflavin (RF) by ATP:riboflavin kinase (RFK) activity at its C-terminal domain. The N-terminal domain converts FMN to FAD under a reducing environment by FMN:ATP adenylyltransferase (FMNAT) activity which is reversible (FAD pyrophosphorylase activity). Herein, we investigated the role of F26 residue of the 24-GFFD-28 motif of SaFADS FMNAT domain, mostly conserved in the reducing agent-dependent FADSs. The steady-state kinetics studies showed changes in the KmATP values for mutants, indicating that the F26 residue is crucial for the FMNAT activity. Further, the FMNAT activity of the F26S mutant was observed to be higher than that of the wild-type SaFADS and its other variants at lower reducing agent concentration. In addition, the FADpp activity was inhibited by an excess of FAD substrate, which was more potent in the mutants. The altered orientation of the F26 side-chain observed in the molecular dynamics analysis suggested its plausible involvement in stabilizing FMN and ATP substrates in their respective binding pockets. Also, the SaFADS ternary complex formed with reduced FMN exhibited significant structural changes in the ß4n-ß5n and L3n regions compared to the oxidised FMN bound and apo forms of SaFADS. Overall, our data suggests the functional role of F26 residue in the FMNAT domain of SaFADS.

The bacterial iron sensor IdeR recognizes its DNA targets by indirect readout.

The iron-dependent regulator IdeR is the main transcriptional regulator controlling iron homeostasis genes in Actinobacteria, including species from the Corynebacterium, Mycobacterium and Streptomyces genera, as well as the erythromycin-producing bacterium Saccharopolyspora erythraea. Despite being a well-studied transcription factor since the identification of the Diphtheria toxin repressor DtxR three decades ago, the details of how IdeR proteins recognize their highly conserved 19-bp DNA target remain to be elucidated. IdeR makes few direct contacts with DNA bases in its target sequence, and we show here that these contacts are not required for target recognition. The results of our structural and mutational studies support a model wherein IdeR mainly uses an indirect readout mechanism, identifying its targets via the sequence-dependent DNA backbone structure rather than through specific contacts with the DNA bases. Furthermore, we show that IdeR efficiently recognizes a shorter palindromic sequence corresponding to a half binding site as compared to the full 19-bp target previously reported, expanding the number of potential target genes controlled by IdeR proteins.

Inhibition of microbial production of the malodorous substance isovaleric acid by 4,4' dichloro 2-hydroxydiphenyl ether (DCPP).

Human body malodour is a complex phenomenon. Several types of sweat glands produce odorless secretions that are metabolized by a consortium of skin-resident microorganisms to a diverse set of malodorous substances. Isovaleric acid, a sweaty-smelling compound, is one major malodorous component produced by staphylococci with the skin-derived amino acid L-leucine as a substrate. During wearing, fabrics are contaminated with sweat and microorganisms and high humidity propagates growth and microbial malodour production. Incomplete removal of sweat residues and microorganisms from fabrics during laundry with bleach-free detergents and at low temperatures elevate the problem of textile malodour. This study aimed to analyze the inhibitory effect of the antimicrobial 4,4' dichloro 2-hydroxydiphenyl ether (DCPP) on the formation of isovaleric acid on fabrics. Therefore, GC-FID- and GC-MS-based methods for the analysis of isovaleric acid in an artificial human sweat-mimicking medium and in textile extracts were established. Here, we show that antimicrobials capable to deposit on fabrics during laundry, such as DCPP, are effective in growth inhibition of typical malodour-generating bacteria and prevent the staphylococcal formation of isovaleric acid on fabrics in a simple experimental setup. This can contribute to increased hygiene for mild laundry care approaches, where bacterial contamination and malodour production represent a considerable consumer problem.

The actinobacterial WhiB-like (Wbl) family of transcription factors.

The WhiB-like (Wbl) family of proteins are exclusively found in Actinobacteria. Wbls have been shown to play key roles in virulence and antibiotic resistance in Mycobacteria and Corynebacteria, reflecting their importance during infection by the human pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Corynebacterium diphtheriae. In the antibiotic-producing Streptomyces, several Wbls have important roles in the regulation of morphological differentiation, including WhiB, a protein that controls the initiation of sporulation septation and the founding member of the Wbl family. In recent years, genome sequencing has revealed the prevalence of Wbl paralogues in species throughout the Actinobacteria. Wbl proteins are small (generally ~80-140 residues) and each contains four invariant cysteine residues that bind an O2 - and NO-sensitive [4Fe-4S] cluster, raising the question as to how they can maintain distinct cellular functions within a given species. Despite their discovery over 25 years ago, the Wbl protein family has largely remained enigmatic. Here I summarise recent research in Mycobacteria, Corynebacteria and Streptomyces that sheds light on the biochemical function of Wbls as transcription factors and as potential sensors of O2 and NO. I suggest that Wbl evolution has created diversity in protein-protein interactions, [4Fe-4S] cluster-sensitivity and the ability to bind DNA.

Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109.

BACKGROUND: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. RESULTS: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum ΔporAΔporH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). CONCLUSIONS: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.

In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking.

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2M synthesizes pilus fibers with correct Lys-Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA-SrtA-SpaA polymerization intermediate depicts SrtA2M sandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2M can terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria

ABSTRACT Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria.

Role of microbes in bioremediation of oil spills has become inevitable owing to their eco friendly nature. This study focused on the isolation and characterization of bacterial strains with superior oil degrading potential from crude-oil contaminated soil. Three such bacterial strains were selected and subsequently identified by 16S rRNA gene sequence analysis as Corynebacterium aurimucosum, Acinetobacter baumannii and Microbacterium hydrocarbonoxydans respectively. The specific activity of catechol 1,2 dioxygenase (C12O) and catechol 2,3 dioxygenase (C23O) was determined in these three strains wherein the activity of C12O was more than that of C23O. Among the three strains, Microbacterium hydrocarbonoxydans exhibited superior crude oil degrading ability as evidenced by its superior growth rate in crude oil enriched medium and enhanced activity of dioxygenases. Also degradation of total petroleum hydrocarbon (TPH) in crude oil was higher with Microbacterium hydrocarbonoxydans. The three strains also produced biosurfactants of glycolipid nature as indicated d by biochemical, FTIR and GCMS analysis. These findings emphasize that such bacterial strains with superior oil degrading capacity may find their potential application in bioremediation of oil spills and conservation of marine and soil ecosystem.

Efficient multi-enzyme-catalyzed CDP-choline production driven by an ATP donor module.

Cytidine diphosphate choline (CDP-choline) has been applied for treating acute craniocerebral injury and allowing recovery of consciousness after brain surgery. In this study, an acetate kinase (ACK)/acetyl phosphate system was used to supply ATP and combined with Escherichia coli-overexpressed CMP kinase (CMK), NDP kinase (NDK), choline phosphate cytidylyltransferase (CCT), and choline kinase (CKI) to produce CDP-choline from CMP and choline chloride. Within 1 h, 49 mM CDP-choline was produced, for a molar yield of 89.9 and 68.4 % based on CMP and choline chloride, respectively; the utilization efficiency of energy (UEE) was 79.5 %. Acetyl phosphate, sodium acetate, and CTP inhibited the reaction when the concentration exceeded 18.5, 600, and 30 mM, respectively. This inhibition could be overcome by controlling the rate of acetyl phosphate, CMP addition or using KOH instead of NaOH to regulate the pH in fed-batch transformation. After 24 h, the maximum titer was 124.1 ± 2.7 mM, the productivity was 5.1 ± 0.1 mM l-1 h-1, the molar yield to CMP and choline chloride were 83.8 and 63.7 %, respectively, and the UEE was 58.2 %. This high yield and productivity of CDP-choline through biocatalysis suggest future application at the industrial scale.

[CORYNEBACTERIUM: FEATURES OF THE STRUCTURE OF THE BACTERIAL CELL].

In a review of the features of the bacterial cells are Corynebacterium structure: characterized by an upper layer, highly organized cell wall, cytoplasmic membrane, cytoplasm, nucleoid. Described in detail the structure of the upper layer containing pili (fimbriae), microcapsule surface proteins - PS-2, DIP1281, 67-72r protein (hemagglutinin), porins, sialidase (neuraminidase). These components are the ability to initiate a serial of Corynebacterium work with the host cell, followed by colonization. It submitted a detailed description .of the structure and functions of cell wall structures - cord factor, which is a second barrier permeability; arabinogalactan, peptidoglycan, lipomannan and lipoarabinomannan. The structure and function of the cytoplasmic membrane as the main diffusion barrier cell cytoplasm and the genome of Corynebacterium. Presented differ- ent molecular genetic methods for the identification and differentiation of closely related species of Corynebacterium.

The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

Urine/Plasma Neutrophil Gelatinase Associated Lipocalin Ratio Is a Sensitive and Specific Marker of Subclinical Acute Kidney Injury in Mice.

BACKGROUND: Detection of acute kidney injury (AKI) is still a challenge if conventional markers of kidney function are within reference range. We studied the sensitivity and specificity of NGAL as an AKI marker at different degrees of renal ischemia. METHODS: Male C57BL/6J mice were subjected to 10-, 20- or 30-min unilateral renal ischemia, to control operation or no operation, and AKI was evaluated 1 day later by histology, immunohistochemistry, BUN, creatinine, NGAL (plasma and urine) and renal NGAL mRNA expression. RESULTS: A short (10-min) ischemia did not alter BUN or kidney histology, but elevated plasma and urinary NGAL level and renal NGAL mRNA expression although to a much smaller extent than longer ischemia. Surprisingly, control operation elevated plasma NGAL and renal NGAL mRNA expression to a similar extent as 10-min ischemia. Further, the ratio of urine to plasma NGAL was the best parameter to differentiate a 10-min ischemic injury from control operation, while it was similar in the non and control-operated groups. CONCLUSIONS: These results suggest that urinary NGAL excretion and especially ratio of urine to plasma NGAL are sensitive and specific markers of subclinical acute kidney injury in mice.

Recognition of Corynebacterium pseudodiphtheriticum by Toll-like receptors and up-regulation of antimicrobial peptides in human corneal epithelial cells.

Bacterial keratitis is a major cause of corneal ulcers in developing and industrialized nations. In this study, we examined the host innate immune responses to Corynebacterium pseudodiphtheriticum, often overlooked as commensal, in human corneal epithelial cells. The expressions of innate immune mediators were determined by quantitative PCR from corneal ulcers of patients and immortalized human corneal epithelial cells (HCEC). We have found an elevated expression of Toll like receptors (TLRs) along with IL-6 and IL-1ß from both ulcers and epithelial cells infected with C. pseudodiphtheriticum. Activation of NF-κB and MAPK signaling pathways were also observed in HCEC in response to C. pseudodiphtheriticum. In addition, we found a significant increase in the expression of antimicrobial peptides S100A8, S100A9 and human ß-defensin 1 from both corneal ulcers and HCEC.

Identification of axillary Staphylococcus sp. involved in the production of the malodorous thioalcohol 3-methyl-3-sufanylhexan-1-ol.

The production of malodour by humans is mediated by bacterial transformation of naturally secreted, non-odorous molecules. Specifically in the underarm (axilla), malodour arises due to biotransformation by the microbiota of dipeptide-conjugated thioalcohols, particularly S-[1-(2-hydroxyethyl)-1-methylbutyl]-(L)-cysteinylglycine (Cys-Gly-3M3SH). This molecule, secreted by the axilla, has a well-established role in malodour when metabolized to free thioalcohol by bacteria. We present Cys-Gly-3M3SH biotransformation data from a library of skin-isolated corynebacteria and staphylococci and report a significant variation in thioalcohol generation across individual bacterial species. Staphylococcus hominis, Staphylococcus haemolyticus and Staphylococcus lugdunensis were particularly efficient Cys-Gly-3M3SH transformers. In contrast, Staphylococcus epidermidis and Corynebacterium tuberculostearicum, both highly prevalent axillary commensals, are low producers of 3M3SH. We also identify significant differences between the ability of several isolates to biotransform Cys-Gly-3M3SH compared to S-benzyl-L-Cys-Gly, a dipeptide-linked version of a commonly used malodour precursor substrate. Finally, using traditional biochemical assays we subsequently establish that Cys-Gly-3M3SH is actively transported into S. hominis, rather than passively diffusing across the membrane. This work significantly enhances our knowledge of Cys-Gly-3M3SH biotransformation by physiologically important bacteria in the axillary microbiota.

Isolation and characterization of crude oil degrading bacteria from the Persian Gulf (Khorramshahr provenance).

Fifteen crude oil degrading bacteria were isolated from oil contaminated sites in the Persian Gulf at Khorramshahr provenance. These bacteria were screened with two important factors such as growth rate on crude oil and hydrocarbon biodegradation, and then three strains were selected from 15 isolated strains for further study. One strain (PG-Z) that show the best crude oil biodegradation was selected between all isolates. Nucleotides sequencing of the gene encoding for 16S rRNA show that strain PG-Z belong to Corynebacterium variabile genus. This strain was efficient in degrading of crude oil. This strain was capable to degraded 82% of crude-oil after one week incubation in ONR7a medium. The PG-Z strain had high emulsification activity and biosurfactant production between all isolates. GC-MS analysis shows that C. variabile strain PG-Z can degrade different alkanes in crude oil.

Utility and pattern of positivity of p40 in the diagnosis of squamous cell carcinoma of the lung by cytology: the first study on fine needle aspiration smears.

OBJECTIVE: Immunohistochemistry for p40 has emerged as a clinically applicable tool with high sensitivity and specificity to distinguish lung adenocarcinoma and squamous cell carcinoma (SCC). It appears to be an excellent marker for squamous differentiation. Although application of p40 in cell blocks has been reported, its expression has not been described in cytology smears. The aim was to study the expression of p40 in fine needle aspirates of SCC of the lung and to analyse differences in immunoreactivity in variably differentiated SCC. METHODS: The study used aspirates of lung masses diagnosed as SCC over a period of 2 years. p40 immunocytochemistry was performed on destained Papanicolaou smears. Nuclear staining was semi-quantitatively evaluated as 0, 1 + , 2 +  and 3 +  based on the percentage positivity of tumour cells and was correlated with differentiation of the tumour. Adequate unmatched histology (50 biopsies) and cytology (25 smears) controls were taken for comparison. RESULTS: A total of 45 cases of primary and five cases of metastatic pulmonary SCC were identified. There were 17 well, 24 moderately and nine poorly differentiated SCC. p40 immunoreactivity was 2-3 +  in all moderate and poorly differentiated tumours, however, negative to 1 +  in all well-differentiated carcinomas (P < 0.0001). CONCLUSION: p40 immunostain is a valuable stain in identifying lung SCCs and works well in aspiration smears. The pattern of positivity varies with the differentiation of the tumour and is seen prominently in higher grade SCC where in practice the need arises for distinguishing them from either poorly differentiated adenocarcinomas or non-small cell carcinoma, not otherwise specified.

Corynebacterium jeikeium jk0268 constitutes for the 40 amino acid long PorACj, which forms a homooligomeric and anion-selective cell wall channel.

Corynebacterium jeikeium, a resident of human skin, is often associated with multidrug resistant nosocomial infections in immunodepressed patients. C. jeikeium K411 belongs to mycolic acid-containing actinomycetes, the mycolata and contains a channel-forming protein as judged from reconstitution experiments with artificial lipid bilayer experiments. The channel-forming protein was present in detergent treated cell walls and in extracts of whole cells using organic solvents. A gene coding for a 40 amino acid long polypeptide possibly responsible for the pore-forming activity was identified in the known genome of C. jeikeium by its similar chromosomal localization to known porH and porA genes of other Corynebacterium strains. The gene jk0268 was expressed in a porin deficient Corynebacterium glutamicum strain. For purification temporarily histidine-tailed or with a GST-tag at the N-terminus, the homogeneous protein caused channel-forming activity with an average conductance of 1.25 nS in 1M KCl identical to the channels formed by the detergent extracts. Zero-current membrane potential measurements of the voltage dependent channel implied selectivity for anions. This preference is according to single-channel analysis caused by some excess of cationic charges located in the channel lumen formed by oligomeric alpha-helical wheels. The channel has a suggested diameter of 1.4 nm as judged from the permeability of different sized hydrated anions using the Renkin correction factor. Surprisingly, the genome of C. jeikeium contained only one gene coding for a cell wall channel of the PorA/PorH type found in other Corynebacterium species. The possible evolutionary relationship between the heterooligomeric channels formed by certain Corynebacterium strains and the homooligomeric pore of C. jeikeium is discussed.

Biodegradation of petroleum hydrocarbons in the presence of nickel and cobalt.

Bioremediation of environments co-contaminated with hydrocarbons and heavy metals often pose a challenge as heavy metals exert toxicity to existing communities of hydrocarbon degraders. Multi-resistant bacterial strains were studied for ability to degrade hydrocarbons in chemically defined media amended with 5.0 mM Ni(2+), and Co(2+). The bacteria, Pseudomonas aeruginosa CA207Ni, Burkholderia cepacia AL96Co, and Corynebacterium kutscheri FL108Hg, utilized crude oil and anthracene without lag phase at specific growth rate spanning 0.3848-0.8259 per day. The bacterial populations grew in hydrocarbon media amended with nickel (Ni) and cobalt (Co) at 0.8393-1.801 days generation time (period of exponential growth, t = 15 days). The bacteria degraded 96.24-98.97, and 92.94-96.24% of crude oil, and anthracene, respectively, within 30 days without any impedance due to metal toxicity (at 5.0 mM). Rather, there was reduction of Ni and Co concentrations in the axenic culture 30 days post-inoculation to 0.08-0.12 and 0.11-0.15 mM, respectively. The metabolic functions of the bacteria are active in the presence of toxic metals (Ni and Co) while utilizing petroleum hydrocarbons for increase in biomass. These findings are useful to other baseline studies on decommissioning of sites co-contaminated with hydrocarbons and toxic metals.