Expression in Pichia pastoris of Thermostable Endo-1,4-ß-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-ß-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-ß-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-ß-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Two ß-glucanases from bacterium Cellulomonas flavigena: expression in Pichia pastoris, properties, biotechnological potential.

In the genome of Cellulomonas flavigena, two genes that potentially encode endoglucanases - Cfla_2912 and Cfla_2913 were identified. We cloned the genes and created Pichia pastoris-based recombinant producers of two proteins that were expressed from the AOX1 promoter. Each of the endoglucanase molecules contains a GH6 catalytic domain, CBM2 carbohydrate-binding module, and TAT signal peptide. The fermentation of the producers was carried out in a 10 L fermenter; Cfla_2912 and Cfla_2913 were purified using affinity chromatography. The yield comprised 10.3 mg/ml (430 U/ml) for Cfla_2913 and 9 mg/ml (370 U/ml) for Cfla_2912. Cfla_2912 and Cfla_2913 were found to have a high activity against barley ß-glucan and lichenan, a weak activity against carboxymethyl cellulose (CMC), phosphoric-acid treated cellulose, and no activity against laminarin, xylan, soluble starch, microcrystalline cellulose, cellobiose, and cellotriose. Thus, the proteins exhibited ß-glucanase activity. Both proteins had a neutral pH optimum of about 7.0 and were more stable at neutral and slightly alkaline pH ranging from 7.0 to 9.0. Cfla_2912 and Cfla_2913 showed a moderate thermal stability. The products of barley ß-glucan hydrolysis by Cfla_2912 and Cfla_2913 were trisaccharide, tetrasaccharide, and cellobiose. Cfla_2912 and Cfla_2913 efficiently hydrolyzed cereal polysaccharides, which indicate that they may have biotechnological potential.

Regulation of gene expression in restriction-modification system Eco29kI.

The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

Troponin elevations following vascular surgery in patients without preoperative myocardial ischemia.

OBJECTIVES: A normal preoperative myocardial perfusion-imaging (MPI) test in advance of vascular surgery predicts a low risk of postoperative clinical events at 30 days. Among patients undergoing vascular surgery with a normal preoperative MPI, cardiac troponin I (cTnI) elevations are common and predictive of a poor long-term outcome. METHODS: The study cohort comprised 182 patients. Between January 2005 and December 2009, we studied these patients, who had no evidence of myocardial ischemia on preoperative MPI and were undergoing vascular surgery. Blood was obtained in all of the patients in the first 2 days following vascular surgery, and cTnI levels were measured. The values that exceeded the upper reference limit (URL) were categorized as either low (+) (greater than or equal to the URL but less than three times the URL) or high (+) (greater than or equal to three times the URL). Long-term survival was determined from the time of the vascular operation. RESULTS: The mean age of the population was 69 ± 8 years, and the mean revised cardiac risk index was 1.80 ± 0.77. The most common indication for vascular intervention was an expanding abdominal aortic aneurysm (n = 96, 52.5%). Within 48 hours of surgery, 58 patients (32%) had a typical rise and fall in TnI, with at least one value exceeding the URL. Of these patients, 17 (9%) were classified as high (+) and 41 (22.5%) as low (+). At 1 year post-vascular surgery, mortality was 8% for the overall cohort. A high (+) Tn elevation was an identifier of decreased 1-year survival (29%) relative to normal (3%) and low (+) (14%; P < 0.001). Stratified cTn was an independent predictor of the long-term risk of death. CONCLUSIONS: Among patients undergoing vascular procedures without evidence of myocardial ischemia on MPI, an elevation in TnI is common and predictive of long-term mortality risk.

Voluntary Control of Cognitive Activity in Preschool Children: Age-dependent Changes from Ages 3-4 to 4-5.

Background: Voluntary control of goal-directed behavior and mental activity in preschool children plays a key role in knowledge acquisition and future academic achievement. Studies of voluntary control have mainly concerned 6-8-year old children; much less is known about the ability to exercise voluntary control at early ages. Due to the high prognostic value of the level of development of voluntary control and heterogeneous development of their individual components, it seems actually useful to study age-related changes of these components in children from 3-4 to 4-5 years old. Objective: To compare age-related changes in executive functions (EF) in children age 3-4 years (mean age: 3.5±0.2 yrs; n = 49; 31 boys) and 4-5 years (mean age: 4.5±0.3 yrs; n = 70; 35 boys). Design: To assess the different components of EF we used: 1) a qualitative group and individual testing procedure based on the principles of Luria's theory of the dynamic localization and organization of higher mental functions; and 2) a computerized testing procedure which included the Bourdon-Wiersma cancellation test, the "Hearts and Flowers" conflict test (the Dots task), and the Corsi block-tapping test. Results: The results showed that different components of voluntary control developed at different rates (heterochronically): there were significant progressive changes from 3-4 to 4-5 years for working memory, assimilation of instructions, switching between separate actions, selective concentration on a target or task, and the distribution of attention. Some other components of EF did not show significant positive dynamics during this period. Conclusion: The results indicate the importance of applying the activity theory approach to the development of cognitive processes in preschool age.

Transcription regulation of restriction-modification system Ecl18kI.

Restriction-modification (R-M) system Ecl18kI is representative of R-M systems whose coordinated transcription is achieved through a separate DNA-binding domain of the methyltransferase. M.Ecl18kI recognizes an operator sequence located in the noncoding region that separates the divergently transcribed R and M genes. Here we show that, contrary to previous predictions, the two ecl18kI promoters are not divergent, but actually face one another. The binding of M.Ecl18kI to its operator prevents RNA polymerase (RNAP) binding to the M promoter by steric exclusion, but has no direct effect on RNAP interaction with the R promoter. The start point for R transcription is located outside of the intergenic region, opposite the initiation codon of the M gene. Regulated transcription of the potentially toxic ecl18kI R gene is accomplished (i) at the stage of promoter complex formation, through direct competition from complexes formed at the M promoter, and (ii) at the stage of promoter clearance, since R promoter-bound RNAP escapes the promoter more slowly than RNAP bound to the M promoter.

Transcription regulation of restriction-modification system Esp1396I.

The convergently transcribed restriction (R) and methylase (M) genes of the Restriction-Modification system Esp1396I are tightly regulated by a controller (C) protein that forms part of the CR operon. We have mapped the transcriptional start sites from each promoter and examined the regulatory role of C.Esp1396I in vivo and in vitro. C-protein binding at the CR and M promoters was analyzed by DNA footprinting and a range of biophysical techniques. The distal and proximal C-protein binding sites at the CR promoter are responsible for activation and repression, respectively. In contrast, a C-protein dimer binds to a single site at the M-promoter to repress the gene, with an affinity much greater than for the CR promoter. Thus, during establishment of the system in a naïve host, the activity of the M promoter is turned off early, preventing excessive synthesis of methylase. Mutational analysis of promoter binding sites reveals that the tetranucleotide inverted repeats long believed to be important for C-protein binding to DNA are less significant than previously thought. Instead, symmetry-related elements outside of these repeats appear to be critical for the interaction and are discussed in terms of the recent crystal structure of C.Esp139I bound to the CR promoter.

The Naphthalene Catabolic Genes of Pseudomonas putida BS3701: Additional Regulatory Control.

Pseudomonas microorganisms are used for bioremediation of soils contaminated with petroleum hydrocarbons. The overall remediation efficiency is largely dependent on the presence of macro- and micronutrients. Widely varying concentrations of available nitrogen and iron (Fe) in soils were shown to affect residual hydrocarbons in the course of biodegradation. The regulatory mechanisms of expression of hydrocarbon catabolic genes in low nitrogen/low iron conditions remain unclear. The catabolism of naphthalene, a two-ring polycyclic aromatic hydrocarbon, has been well studied in pseudomonads in terms of the involvement of specific transcriptional activators, thus making it useful in revealing additional regulatory control of the adaptation of hydrocarbon destructors to a low level of the essential nutrients. The Pseudomonas putida strain BS3701 is a component of the "MicroBak" preparation for soil remediation. Previously, this strain was shown to contain genes encoding the key enzymes for naphthalene catabolism: naphthalene 1,2-dioxygenase, salicylate hydroxylase, catechol 2,3-dioxygenase, and catechol 1,2-dioxygenase. Our study aimed to clarify whether the naphthalene catabolic gene expression is dependent on the amount of nitrogen and iron in the growth culture medium, and if so, at exactly which stages the expression is regulated. We cultivated the strain in low nitrogen/low iron conditions with the concurrent evaluation of the activity of the key enzymes and the mRNA level of genes encoding these enzymes. We are the first to report that naphthalene catabolic genes are subject not only to transcriptional but also post-transcriptional regulation.

Complete Genome Sequence of Pseudomonas putida BS3701, a Promising Polycyclic Aromatic Hydrocarbon-Degrading Strain for Bioremediation Technologies.

The strain Pseudomonas putida BS3701 was isolated from soil contaminated with coke by-product waste (Moscow Region, Russian Federation). It is capable of degrading crude oil and polycyclic aromatic hydrocarbons (PAHs). The P. putida BS3701 genome consists of a 6,337,358-bp circular chromosome and two circular plasmids (pBS1141 with 107,388 bp and pBS1142 with 54,501 bp).

An alternative approach to study the enzymatic specificities of the CfrBI restriction-modification system.

Restriction-modification systems (RMS) are the main gene-engineering tools and a suitable model to study the molecular mechanisms of catalysis and DNA-protein interactions. Research into the catalytic properties of these enzymes, determination of hydrolysis and DNA-methylation sites remain topical. In our previous work we have cloned and sequenced the CfrBI restriction-modification system (strain Citrobacter freundii), which recognizes the nucleotide sequence 5'-CCWWGG-3'. In this article we describe the cloning of the methyltransferase and restriction endonuclease genes (gene encoding CfrBI DNA methyltransferase (cfrBIM) and gene encoding CfrBI restriction endonuclease (cfrBIR)) separately to obtain strains overproducing the enzymes of this system. His6-CfrBI, which had been purified to homogeneity, was used to establish the DNA-hydrolysis point in its recognition site. CfrBI was shown to cleave DNA after just the first 5'C within the recognition site and then to generate 4-nt 3' cohesive ends (5'-C/CWWGG-3'). To map the site of methylation by M.CfrBI, we exploited the fact that the CfrBI site partially overlaps with the recognition sites of the well-documented enzymes KpnI and ApaI. The M.CfrBI- induced hemimethylation of the internal C residue of the ApaI recognition sequence (GGGCN4mCC) was observed to block cleavage by ApaI. In contrast, KpnI was able to digest its M.CfrBI-hemimethylated site (GGTAN4mCC). KpnI was used to restrict a fragment of DNA harbouring the CfrBI and KpnI sites, in which the CfrBI site was methylated in vitro by His6-M.CfrBI using [3H]-SAM. The subsequent separation of hydrolysis products by electrophoresis and the enumeration of incorporated [H3]-methyl groups in each of the fragments made it possible to determine that external cytosine undergoes modification in the recognition site.

Type II restriction endonuclease R.Eco29kI is a member of the GIY-YIG nuclease superfamily.

BACKGROUND: The majority of experimentally determined crystal structures of Type II restriction endonucleases (REases) exhibit a common PD-(D/E)XK fold. Crystal structures have been also determined for single representatives of two other folds: PLD (R.BfiI) and half-pipe (R.PabI), and bioinformatics analyses supported by mutagenesis suggested that some REases belong to the HNH fold. Our previous bioinformatic analysis suggested that REase R.Eco29kI shares sequence similarities with one more unrelated nuclease superfamily, GIY-YIG, however so far no experimental data were available to support this prediction. The determination of a crystal structure of the GIY-YIG domain of homing endonuclease I-TevI provided a template for modeling of R.Eco29kI and prompted us to validate the model experimentally. RESULTS: Using protein fold-recognition methods we generated a new alignment between R.Eco29kI and I-TevI, which suggested a reassignment of one of the putative catalytic residues. A theoretical model of R.Eco29kI was constructed to illustrate its predicted three-dimensional fold and organization of the active site, comprising amino acid residues Y49, Y76, R104, H108, E142, and N154. A series of mutants was constructed to generate amino acid substitutions of selected residues (Y49A, R104A, H108F, E142A and N154L) and the mutant proteins were examined for their ability to bind the DNA containing the Eco29kI site 5'-CCGCGG-3' and to catalyze the cleavage reaction. Experimental data reveal that residues Y49, R104, E142, H108, and N154 are important for the nuclease activity of R.Eco29kI, while H108 and N154 are also important for specific DNA binding by this enzyme. CONCLUSION: Substitutions of residues Y49, R104, H108, E142 and N154 predicted by the model to be a part of the active site lead to mutant proteins with strong defects in the REase activity. These results are in very good agreement with the structural model presented in this work and with our prediction that R.Eco29kI belongs to the GIY-YIG superfamily of nucleases. Our study provides the first experimental evidence for a Type IIP REase that does not belong to the PD-(D/E)XK or HNH superfamilies of nucleases, and is instead a member of the unrelated GIY-YIG superfamily.

Transcription regulation of the EcoRV restriction-modification system.

When a plasmid containing restriction-modification (R-M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R-M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R-M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.

Protein NCRII-18: the role of gene fusion in the molecular evolution of restriction endonucleases.

This work first constructed the fusion protein NCRII-18 by fusing the restriction endonuclease Ecl18kI gene and part of the gene coding for the N-terminal domain of the endonuclease EcoRII. The fusion of the EcoRII N-terminal domain leads to a change in the properties of the recombinant protein. Unlike Ecl18kI, which made the basis of NCRII-18, the fusion protein predominantly recognizes the CCWGG sites, having lost the capability of interacting with the CCSGG sites. Experimental data support the hypothesis of a close evolutionary relationship between type IIE and IIP restriction endonucleases via a recombination between domains with active site structure and elements for recognition with domains responsible for recognition of DNA sequences.

Protein SgpR of Pseudomonas putida strain AK5 is a LysR-type regulator of salicylate degradation through gentisate.

Pseudomonas putida strain AK5 was the first characterized natural strain containing the 'classical' nah1 operon and nahR gene along with genes whose products are responsible for the less explored pathway of salicylate degradation through gentisate (the sgp operon). The sgp operon was found to be preceded by the divergently directed sgpR gene. The amino acid sequence of the sgpR product qualifies it as a LysR-type transcriptional regulator (LTTR) and suggests its potential function as an sgp operon transcriptional regulator. This study focused on verification of SgpR's involvement in regulation of transcription of the operon genes and characterization of its interaction with the sgp promoter. We determined the transcription start for sgpAIKGHB and identified the SgpR binding site. The equilibrium dissociation constant (KD) of the SgpR-DNA complex determined in the presence and absence of the inducer salicylate appeared to be, on the whole, at the lower end of the range for KD values reported for LTTRs. RT-qPCR showed that in the presence of salicylate, efficiency of transcription of the sgpAIKGHB operon increased by three orders of magnitude and reached the highest values so far observed for LTTR-controlled operons, thus holding much promise for further studies of the mechanism of transcriptional regulation that involves SgpR.

Xylanases of Cellulomonas flavigena: expression, biochemical characterization, and biotechnological potential.

Four xylanases of Cellulomonas flavigena were cloned, expressed in Escherichia coli and purified. Three enzymes (CFXyl1, CFXyl2, and CFXyl4) were from the GH10 family, while CFXyl3 was from the GH11 family. The enzymes possessed moderate temperature stability and a neutral pH optimum. The enzymes were more stable at alkaline pH values. CFXyl1 and CFXyl2 hydrolyzed xylan to form xylobiose, xylotriose, xylohexaose, xylopentaose, and xylose, which is typical for GH10. CFXyl3 (GH11) and CFXyl4 (GH10) formed the same xylooligosaccharides, but xylose was formed in small amounts. The xylanases made efficient saccharification of rye, wheat and oat, common components of animal feed, which indicates their high biotechnological potential.

Frequency of Increase in Cardiac Troponin Levels After Peripheral Arterial Operations (Carotid Endarterectomy, Abdominal Aorta Procedure, Distal Bypass) and Their Effect on Medical Management.

The utility of measuring cardiac troponins (cTn) in asymptomatic patients during the perioperative period has been controversial. In the present substudy of the Cardiac Remote Ischemic Preconditioning Prior to Elective Vascular Surgery Trial (NCT01558596), we hypothesized that surveillance of myocardial injury with cTnI in the perioperative period would lead to initiation or intensification of medical therapies for coronary artery disease. Increases in cTnI ≥0.01 µg/l in the perioperative period were considered clinically significant. Intensification of medical therapy was defined as initiation of aspirin or initiation or increases in the dose of angiotensin-converting-enzyme inhibitors or angiotensin-receptor blockers, statins, or ß blockers and was left to the discretion of treating physicians. From June 2011 to April 2015, a total of 185 patients (mean age 68 ± 7 years, 100% men) were enrolled in the trial. A total of 28 patients (15%) had significant increases in cTnI after vascular surgery, and 38 (20.5%) had their medical therapies intensified in the perioperative period. Among patients with increases in cTnI, 11 (39%) had intensification of medical therapy versus 27 patients (17%) with no or smaller increases in cTnI (p = 0.02). Among those patients with ΔcTnI ≥0.01 µg/l, hospital readmissions at 3 to 6 months were 7.6% for the intensification group versus 25% for the no intensification group (p = 0.18). Mortality rate at 6 months was low in both groups (2.6% vs 0%, respectively, p = 0.13). In conclusion, among patients undergoing vascular surgery, perioperative increases in cTn were associated with initiation or intensification of medical therapies for coronary artery disease at the time of discharge.

Cardiac Remote Ischemic Preconditioning Prior to Elective Vascular Surgery (CRIPES): A Prospective, Randomized, Sham-Controlled Phase II Clinical Trial.

BACKGROUND: Remote ischemic preconditioning (RIPC) has been shown to reduce infarct size in animal models. We hypothesized that RIPC before an elective vascular operation would reduce the incidence and amount of a postoperative rise of the cardiac troponin level. METHODS AND RESULTS: Cardiac Remote Ischemic Preconditioning Prior to Elective Vascular Surgery (CRIPES) was a prospective, randomized, sham-controlled phase 2 trial using RIPC before elective vascular procedures. The RIPC protocol consisted of 3 cycles of 5-minute forearm ischemia followed by 5 minutes of reperfusion. The primary endpoint was the proportion of subjects with a detectable increase in cardiac troponin I (cTnI) and the distribution of such increases. From June 2011 to September 2015, 201 male patients (69±7, years) were randomized to either RIPC (n=100) or a sham procedure (n=101). Indications for vascular surgery included an expanding abdominal aortic aneurysm (n=115), occlusive peripheral arterial disease of the lower extremities (n=37), or internal carotid artery stenosis (n=49). Of the 201 patients, 47 (23.5%) had an increase in cTnI above the upper reference limit within 72 hours of the vascular operation, with no statistically significant difference between those patients assigned to RIPC (n=22; 22.2%) versus sham procedure (n=25; 24.7%; P=0.67). Among the cohort with increased cTnI, the median peak values (interquartile range) in the RIPC and control group were 0.048 (0.004-0.174) and 0.017 (0.003-0.105), respectively (P=0.54). CONCLUSIONS: In this randomized, controlled trial of men with increased perioperative cardiac risks, elevation in cardiac troponins was common following vascular surgery, but was not reduced by a strategy of RIPC. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01558596.

Regulation of RNA polymerase promoter selectivity by covalent modification of DNA.

Expression of genes encoding type II restriction/modification (R/M) systems, which are widely spread in eubacteria, must be tightly regulated to ensure that host DNA is protected from restriction endonucleases at all times. Examples of coordinated expression of R/M genes that rely on the action of regulatory factors or the ability of methyl transferases to repress their own synthesis by interacting with the promoter DNA have been described. Here, we characterize the molecular mechanism of factor-independent regulation in the CfrBI R/M system. Regulation of the cfrBIM gene transcription occurs through CfrBIM-catalyzed methylation of a cytosine residue in the cfrBIM promoter. The covalent modification inhibits cfrB1M promoter complex formation by interfering with the RNA polymerase sigma(70) subunit region 4.2 recognition of the -35 promoter element. The decrease in the cfrBIM promoter complex formation leads to increase in the activity of overlapping cfrBIR promoters. This elegant factor-independent regulatory system ensures coordinated expression of the cfrBI genes.

Crystallization and X-ray diffraction studies of a two-domain laccase from Streptomyces griseoflavus.

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases; it is found in many organisms and catalyzes the oxidation of primarily phenolic compounds by oxygen. Two-domain laccases have unusual thermostability, resistance to inhibitors and an alkaline optimum of activity. The causes of these properties in two-domain laccases are poorly understood. A recombinant two-domain laccase (SgfSL) was cloned from the genome of Streptomyces griseoflavus Ac-993, expressed in Escherichia coli and purified to homogeneity. The crystals of SgfSL belonged to the monoclinic space group P21, with unit-cell parameters a = 74.64, b = 94.72, c = 117.40 Å, ß = 90.672°, and diffraction data were collected to 2.0 Šresolution using a synchrotron-radiation source. Two functional trimers per asymmetric unit correspond to a Matthews coefficient of 1.99 Å(3) Da(-1) according to the monomer molecular weight of 35.6 kDa.

Recent Origin of the Methacrylate Redox System in Geobacter sulfurreducens AM-1 through Horizontal Gene Transfer.

The origin and evolution of novel biochemical functions remains one of the key questions in molecular evolution. We study recently emerged methacrylate reductase function that is thought to have emerged in the last century and reported in Geobacter sulfurreducens strain AM-1. We report the sequence and study the evolution of the operon coding for the flavin-containing methacrylate reductase (Mrd) and tetraheme cytochrome с (Mcc) in the genome of G. sulfurreducens AM-1. Different types of signal peptides in functionally interlinked proteins Mrd and Mcc suggest a possible complex mechanism of biogenesis for chromoproteids of the methacrylate redox system. The homologs of the Mrd and Mcc sequence found in δ-Proteobacteria and Deferribacteres are also organized into an operon and their phylogenetic distribution suggested that these two genes tend to be horizontally transferred together. Specifically, the mrd and mcc genes from G. sulfurreducens AM-1 are not monophyletic with any of the homologs found in other Geobacter genomes. The acquisition of methacrylate reductase function by G. sulfurreducens AM-1 appears linked to a horizontal gene transfer event. However, the new function of the products of mrd and mcc may have evolved either prior or subsequent to their acquisition by G. sulfurreducens AM-1.