Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1.

In an effort to improve the understanding of electron transfer mechanisms at the microbe-mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe(3)(PO(4))(2)x8H(2)O] and a switzerite-like phase [Mn(3),Fe(3)(PO(4))(2)x7H(2)O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.

Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1.

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

Oxygen-dependent autoaggregation in Shewanella oneidensis MR-1.

In aerobic chemostat cultures maintained at 50% dissolved O(2) tension (3.5 mg l(-1) dissolved O(2)), Shewanella oneidensis strain MR-1 rapidly aggregated upon addition of 0.68 mM CaCl(2) and retained this multicellular phenotype at high dilution rates. Confocal microscopy analysis of the extracellular matrix material contributing to the stability of the aggregate structures revealed the presence of extracellular DNA, protein and glycoconjugates. Upon onset of O(2)-limited growth (dissolved O(2) below detection) however, the Ca(2+)-supplemented chemostat cultures of strain MR-1 rapidly disaggregated and grew as motile dispersed cells. Global transcriptome analysis comparing aerobic aggregated to O(2)-limited unaggregated cells identified genes encoding cell-to-cell and cell-to-surface adhesion factors whose transcription increased upon exposure to increased O(2) concentrations. The aerobic aggregated cells also revealed increased expression of putative anaerobic electron transfer and homologues of metal reduction genes, including mtrD (SO1782), mtrE (SO1781) and mtrF (SO1780). Our data indicate that mechanisms involved in autoaggregation of MR-1 are dependent on the function of pilD gene which encodes a putative prepilin peptidase. Mutants of S. oneidensis strain MR-1 deficient in PilD and associated pathways, including type IV and Msh pili biogenesis, displayed a moderate increase in sensitivity to H(2)O(2). Taken together, our evidence indicates that aggregate formation in S. oneidensis MR-1 may serve as an alternative or an addition to biochemical detoxification to reduce the oxidative stress associated with production of reactive oxygen species during aerobic metabolism while facilitating the development of hypoxic conditions within the aggregate interior.

Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon.

A modified mariner transposon, miniHimar RB1, was generated to mutagenize cells of the metal-reducing bacterium Shewanella oneidensis. The use of this transposon led to the isolation of stable mutants and allowed rapid identification of disrupted genes. Fifty-eight mutants, including BG104 and BG148 with transposon insertions in the cytochrome c maturation genes ccmC and ccmF1, respectively, were analyzed. Both mutants were deficient in anaerobic respiration and cytochrome c production.

MtrC, an outer membrane decahaem c cytochrome required for metal reduction in Shewanella putrefaciens MR-1.

Shewanella putrefaciens is a facultative anaerobe that can use metal oxides as terminal electron acceptors during anaerobic respiration. Two proteins, MtrB and Cct, have been identified that are specifically involved in metal reduction. Analysis of S. putrefaciens mutants deficient in metal reduction led to the identification of two additional proteins that are involved in this process. MtrA is a periplasmic decahaem c-type cytochrome that appears to be part of the electron transport chain, which leads to Fe(III) and Mn(IV) reduction. MtrC is an outer membrane decahaem c-type cytochrome that appears to be required for the activity of the terminal Fe(III) reductase. Membrane fractions of mutants deficient in MtrC exhibited a decreased level of Fe(III) reduction compared with the wild type. We suggest that MtrC may be a component of the terminal reductase or may be required for its assembly.

Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov.

The genus Shewanella has been studied since 1931 with regard to a variety of topics of relevance to both applied and environmental microbiology. Recent years have seen the introduction of a large number of new Shewanella-like isolates, necessitating a coordinated review of the genus. In this work, the phylogenetic relationships among known shewanellae were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. This polyphasic taxonomy takes into account all available phenotypic and genotypic data and integrates them into a consensus classification. Based on information generated from this study and obtained from the literature, a scheme for the identification of Shewanella species has been compiled. Key phenotypic characteristics were sulfur reduction and halophilicity. Fatty acid and quinone profiling were used to impart an additional layer of information. Molecular characterizations employing small-subunit 16S rDNA sequences were at the limits of resolution for the differentiation of species in some cases. As a result, DNA-DNA hybridization and sequence analyses of a more rapidly evolving molecule (gyrB gene) were performed. Species-specific PCR probes were designed for the gyrB gene and used for the rapid screening of closely related strains. With this polyphasic approach, in addition to the ten described Shewanella species, two new species, Shewanella oneidensis and 'Shewanella pealeana', were recognized; Shewanella oneidensis sp. nov. is described here for the first time.

Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

Anaerobic electron acceptor chemotaxis in Shewanella putrefaciens.

Shewanella putrefaciens MR-1 can grow either aerobically or anaerobically at the expense of many different electron acceptors and is often found in abundance at redox interfaces in nature. Such redox interfaces are often characterized by very strong gradients of electron acceptors resulting from rapid microbial metabolism. The coincidence of S. putrefaciens abundance with environmental gradients prompted an examination of the ability of MR-1 to sense and respond to electron acceptor gradients in the laboratory. In these experiments, taxis to the majority of the electron acceptors that S. putrefaciens utilizes for anaerobic growth was seen. All anaerobic electron acceptor taxis was eliminated by the presence of oxygen, nitrate, nitrite, elemental sulfur, or dimethyl sulfoxide, even though taxis to the latter was very weak and nitrate and nitrite respiration was normal in the presence of dimethyl sulfoxide. Studies with respiratory mutants of MR-1 revealed that several electron acceptors that could not be used for anaerobic growth nevertheless elicited normal anaerobic taxis. Mutant M56, which was unable to respire nitrite, showed normal taxis to nitrite, as well as the inhibition of taxis to other electron acceptors by nitrite. These results indicate that electron acceptor taxis in S. putrefaciens does not conform to the paradigm established for Escherichia coli and several other bacteria. Carbon chemo-taxis was also unusual in this organism: of all carbon compounds tested, the only positive response observed was to formate under anaerobic conditions.

Iron and manganese in anaerobic respiration: environmental significance, physiology, and regulation.

Dissimilatory iron and/or manganese reduction is known to occur in several organisms, including anaerobic sulfur-reducing organisms such as Geobacter metallireducens or Desulfuromonas acetoxidans, and facultative aerobes such as Shewanella putrefaciens. These bacteria couple both carbon oxidation and growth to the reduction of these metals, and inhibitor and competition experiments suggest that Mn(IV) and Fe(III) are efficient electron acceptors similar to nitrate in redox abilities and capable of out-competing electron acceptors of lower potential, such as sulfate (sulfate reduction) or CO2 (methanogenesis). Field studies of iron and/or manganese reduction suggest that organisms with such metabolic abilities play important roles in coupling the oxidation of organic carbon to metal reduction under anaerobic conditions. Because both iron and manganese oxides are solids or colloids, they tend to settle downward in aquatic environments, providing a physical mechanism for the movement of oxidizing potential into anoxic zones. The resulting biogeochemical metal cycles have a strong impact on many other elements including carbon, sulfur, phosphorous, and trace metals.

Sequence and genetic characterization of etrA, an fnr analog that regulates anaerobic respiration in Shewanella putrefaciens MR-1.

An electron transport regulatory gene, etrA, has been isolated and characterized from the obligate respiratory bacterium Shewanella putrefaciens MR-1. The deduced amino acid sequence of etrA (EtrA) shows a high degree of identity to both the Fnr of Escherichia coli (73.6%) and the analogous protein (ANR) of Pseudomonas aeruginosa (50.8%). The four active cysteine residues of Fnr are conserved in EtrA, and the amino acid sequence of the DNA-binding domains of the two proteins are identical. Further, S. putrefaciens etrA is able to complement an fnr mutant of E. coli. In contrast to fnr, there is no recognizable Fnr box upstream of the etrA sequence. Gene replacement etrA mutants of MR-1 were deficient in growth on nitrite, thiosulfate, sulfite, trimethylamine-N-oxide, dimethyl sulfoxide, Fe(III), and fumarate, suggesting that EtrA is involved in the regulation of the corresponding reductase genes. However, the mutants were all positive for reduction of and growth on nitrate and Mn(IV), indicating that EtrA is not involved in the regulation of these two systems. Southern blots of S. putrefaciens DNA with use of etrA as a probe revealed the expected etrA bands and a second set of hybridization signals whose genetic and functional properties remain to be determined.

Differential regulation of insect globin and actin mRNAs during larval development in Chironomus thummi.

S1 nuclease protection assays were used to measure changes in the steady-state levels of six different globin (Gb) mRNAs in the midge, Chironomus thummi thummi (C. thummi, Diptera) during larval development. Two distinct patterns of change were observed. GbI, IV, VIIB-4 and VIIB-5 transcripts were present in 3rd instar larvae, rose from low levels immediately post-moult to peak levels by day 2-3 of the 4th instar, and then declined, reaching near-basal levels by day 7-8. In contrast, transcripts of GbIII (known from previous studies to be 4th instar-specific) and VI, which were undetectable in the 3rd instar, rose to high levels by day 2 of the 4th instar, but remained elevated thereafter. Our data further showed that closely linked Gb genes were not necessarily expressed in a coordinate manner. Unlike the Gb mRNAs, actin (Act) mRNA levels (measured by slot-blot hybridization to a heterologous probe) increased progressively as a proportion of total RNA during 4th instar development. Therefore, the regulation of C. thummi Gb transcript levels is specific, differing from that of Act and among the Gb mRNAs themselves. Elevated 20-hydroxyecdysone (HE) titer at the 3rd-4th instar moult correlates with the low steady-state levels of Gb mRNAs immediately post-moult. However, other aspects of Gb mRNA profiles cannot be explained on the basis of a direct repressive effect by HE on Gb gene transcription.

Multiple clustered genes of the haemoglobin VIIB subfamily of Chironomus thummi thummi (Diptera).

The nucleotide and inferred amino acid sequence of three globin VIIB genes from the midge, Chironomus thummi thummi have been determined. These three genes are intronless, like all previously reported Chironomid globin genes. Transcription of globin genes VIIB-4, VIIB-5 (and/or VIIB-6) and VIIB-7 was demonstrated by S1 nuclease protection analysis. Comparison of actual and inferred globin VIIB amino acid sequences reveals that (i) most of the amino acid substitutions are conservative in nature, and (ii) none of the substitutions are expected to interfere with the ability of the globins to bind haem. Nucleotide sequence comparisons suggest that gene duplications and partial gene corrections (conversions) have occurred recently in the globin VIIB subfamily locus.

Deoxynucleotide sequence of an insect cDNA codes for an unreported member of the Chironomus thummi globin family.

Synthetic oligonucleotides served as probes to isolate insect globin clones from a Chironomus thummi cDNA bank. The cDNA insert of one clone (pC-S9) was completely sequenced by the dideoxy termination procedure. Beginning at the 5' end of the coding region, the 584 base pair sequence encodes most of an N-terminal hydrophobic signal sequence and the complete sequence for a mature secreted globin, and contains a polyadenylation recognition site 3' to an appropriate stop codon. The inferred amino acid sequence is that of an unreported variant of hemoglobin VIIB. Based on the number of differences between Hb VIIB chains, the pC-S9 gene has been evolutionarily independent longer than the other (two) members of the globin VIIB subfamily.