Escherichia coli ClpX, a member of the Clp family of ATPases, has ATP-dependent chaperone activity and is required for specific ATP-dependent proteolytic activities expressed by ClpP. Gel filtration and electron microscopy showed that ClpX subunits (M r 46,000) associate to form a six-membered ring (M r ϳ 280,000) that is stabilized by binding of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of two seven-membered rings stacked face-to-face, interacts with the nucleotide-stabilized hexamer of ClpX to form a complex that could be isolated by gel filtration. Electron micrographs of negatively stained ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in which ClpP was flanked on either one or both sides by a ring of ClpX. Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding interactions between the seven-membered rings of ClpP and the six-membered rings of ClpX. Competition studies showed that ClpA may have a slightly higher affinity (ϳ2-fold) for binding to ClpP. Mixed complexes of ClpA, ClpX, and ClpP with the two ATPases bound simultaneously to opposite faces of a single ClpP molecule were seen by electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against oligopeptide substrates (>10,000 min ؊1

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Oxidation of methionine residues to methionine sulfoxide can lead to inactivation of proteins. Methionine sulfoxide reductase (MsrA) has been known for a long time, and its repairing function well characterized. Here we identify a new methionine sulfoxide reductase, which we referred to as MsrB, the gene of which is present in genomes of eubacteria, archaebacteria, and eucaryotes. The msrA and msrB genes exhibit no sequence similarity and, in some genomes, are fused. The Escherichia coli MsrB protein (currently predicted to be encoded by an open reading frame of unknown function named yeaA) was used for genetic, enzymatic, and mass spectrometric investigations. Our in vivo study revealed that msrB is required for cadmium resistance of E. coli, a carcinogenic compound that induces oxidative stress. Our in vitro studies, showed that (i) MsrB and MsrA enzymes reduce free methionine sulfoxide with turnover rates of 0.6 min ؊1 and 20 min ؊1 , respectively, (ii) MsrA and MsrB act on oxidized calmodulin, each by repairing four to six of the eight methionine sulfoxide residues initially present, and (iii) simultaneous action of both MsrA and MsrB allowed full reduction of oxidized calmodulin. A possibility is that these two ubiquitous methionine sulfoxide reductases exhibit different substrate specificity.

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A laser ablation-ICPMS method using an infrared (1030 nm), low-energy (39 microJ/pulse), high repetition rate (10 kHz), femtosecond laser was developed to improve the sensitivity of detection of heteroatom-containing proteins in 1D polyacrylamide gels. A 2-mm-wide lane was ablated by ultrafast (10 cm s(-1)) back-and-forth movement of a 20-microm laser beam parallel to the protein bands while the gel advanced perpendicularly. This procedure resulted in a considerable increase in detection sensitivity (>40-fold) compared to the nanosecond 266-nm laser ablation-ICPMS, mainly because of the much larger amount of ablated material introduced into the plasma on the time scale of the dwell time of the mass spectrometer. The method was applied to the specific detection in the gel of formate dehydrogenase expressed in Escherichia coli and of selenoproteins in Desulfococcus multivorans with detection limits at the low-femtomolar levels.

Desulfitobacterium spp. are ubiquitous organisms with a broad metabolic versatility, and some isolates have the ability to use tetrachloroethene (PCE) as terminal electron acceptor. In order to identify proteins involved in this organohalide respiration process, a comparative proteomic analysis was performed. Soluble and membrane-associated proteins obtained from cells of Desulfitobacterium hafniense strain TCE1 that were growing on different combinations of the electron donors lactate and hydrogen and the electron acceptors PCE and fumarate were analyzed. Among proteins increasingly expressed in the presence of PCE compared to fumarate as electron acceptor, a total of 57 proteins were identified by mass spectrometry analysis, revealing proteins involved in stress response and associated regulation pathways, such as PspA, GroEL, and CodY, and also proteins potentially participating in carbon and energy metabolism, such as proteins of the WoodLjungdahl pathway and electron transfer flavoproteins. These proteomic results suggest that D. hafniense strain TCE1 adapts its physiology to face the relative unfavorable growth conditions during an apparent opportunistic organohalide respiration.The first members of the genus Desulfitobacterium have been isolated as organohalide-respiring bacteria able to use chlorinated aliphatic (chloroethenes and -ethanes) and/or aromatic compounds as terminal electron acceptor (6,19,39). The variety of environments from which Desulfitobacterium strains have been isolated suggests that they are ubiquitous organisms (for a review, see reference 34). Their ability to use such toxic chlorinated compounds as electron acceptors has probably allowed them to colonize a number of particular environmental niches. However, they survive probably thanks to their metabolic versatility using various nonchlorinated electron acceptors, such as fumarate, nitrate, sulfite, thiosulfate, humic acids, and metals (reviewed in reference 39).The recently sequenced genomes of Desulfitobacterium hafniense strains Y51 (NCBI reference strain NC_007907) (23) and DCB-2 (NCBI reference strain NC_011830) (DOE Joint Genome Institute) have unraveled additional aspects of the metabolic versatility of this genus. A total of 59 members of the conserved iron-sulfur molybdoenzyme (CISM) family (28), previously known as the dimethyl sulfoxide (DMSO) reductase family, have been identified in the genome of strain Y51, among which are enzymes with a possible role in anaerobic respiration of DMSO, trimethylamine N-oxide, polysulfide, selenate, and arsenate. This genome also contains 18 paralogues of the fumarate reductase, indicating a gene reservoir far beyond the recognized physiological capabilities of Desulfitobacterium spp., especially with respect to their already considerable respiratory flexibility.Anaerobic respiration implies the establishment of a structured electron transport chain within the cytoplasmic membrane enabling energy conservation via the proton motive force, and the electron transport chains can sometimes ...

Bacteriophage Mu repressor, which is stable in its wildtype form, can mutate to become sensitive to its Escherichia coli host ATP‐dependent ClpXP protease. We further investigated the determinants of the mutant repressor's sensitivity to Clp. We show the crucial importance of a C‐terminal, seven amino acid long sequence in which a single change is sufficient to decrease the rate of degradation of the protein. The sequence was fused at the C‐terminal end of the CcdB and CcdA proteins encoded by plasmid F. CcdB, which is naturally stable, was unaffected, while CcdA, which is normally degraded by the Lon protease, became a substrate for ClpXP while remaining a substrate for Lon. In agreement with the current hypothesis on the mechanism of recognition of their substrates by energy‐ dependent proteases, these results support the existence, on the substrate polypeptides, of separate motifs responsible for recognition and cleavage by the protease.

f Marinobacter hydrocarbonoclasticus SP17 forms biofilms specifically at the interface between water and hydrophobic organic compounds (HOCs) that are used as carbon and energy sources. Biofilm formation at the HOC-water interface has been recognized as a strategy to overcome the low availability of these nearly water-insoluble substrates. Here, we present the genome sequence of SP17, which could provide further insights into the mechanisms of enhancement of HOCs assimilation through biofilm formation.H ydrophobic organic compounds (HOCs) encompassing lipids, hydrocarbons, and some organic pollutants are widely distributed in the environment but are weakly soluble in water and as a consequence poorly available for assimilation by heterotrophic bacteria. Biofilm formation at the HOC-water interface is a strategy employed by Marinobacter hydrocarbonoclasticus SP17 (ATCC 49840) to overcome the low bioavailability of HOCs. SP17 was isolated from chronically oil-contaminated sediment for its ability to use alkanes as the sole carbon and energy source (5). M. hydrocarbonoclasticus is a Gram-negative, aerobic, motile, nonspore-forming, and rod-shaped bacterium (5). It exhibits extreme halotolerance (0.08 to 3.5 M NaCl) and synthesizes ectoine as an osmoprotectant (2, 3).SP17 adheres and forms biofilms on alkanes and produces an extracellular-surface-active compound (2, 6). Physiological and proteomic studies revealed that biofilm formation is an efficient strategy to colonize hydrophobic interfaces (1,11,12). SP17 forms biofilms at the interface between aqueous-phase and HOC substrates like n-alkanes, fatty alcohols, or apolar lipids, such as wax esters and triacylglycerols. In contrast, biofilms were not observed on the nonmetabolizable compounds (n-C 32 alkanes, pristane, and heptamethylnonane) and glass or plastics (7). The discrimination between metabolizable and nonmetabolizable compounds indicates that at some level, biofilm formation is controlled by the presence of a nutritive interface. Adhesion and biofilm formation could be a behavioral strategy to acquire carbon and energy from HOCs contained in marine aggregates.The sequencing of the M. hydrocarbonoclasticus SP17 genome was obtained using a conventional whole-genome shotgun strategy with three libraries (3-, 10-, and 25-kb fragments) on ABI3730 sequencers. Assembly was done using the Phred/Phrap/Consed software package (www.phrap.org) with primer walking, PCR, and in vitro transposition technology (Template generation system II kit; Finnzyme, Espoo, Finland) as finishing steps, yielding a single contig molecule without gaps. Automatic genome annotation was performed using the MAGE annotation server (9, 10) followed by manual annotation. The genome of SP17 encompasses a unique chromosome with a similar GϩC content (57.43%) to but a smaller size (3989,480 bases) than that of the other Marinobacter genomes (ranging between 4,333 and 4,894 kb) (4, 8, 13). The SP17 genome contains 3 rRNA operons, 50 tRNA genes, and 3807 protein-coding sequences (CDSs) (967.89-b...