SummaryThroughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a threedimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.

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We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H 2 , or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (␣-FeOOH) forms, and above pH 7.2 magnetite (Fe 3 O 4 ) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ϳ10 ؊5 . To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B 12 ) biosynthesis.

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels

Interactions between microorganisms shape microbial ecosystems. Systematic studies of mixed microbes in co-culture have revealed widespread potential for growth inhibition among marine heterotrophic bacteria, but similar synoptic studies have not been done with autotroph/heterotroph pairs, nor have precise descriptions of the temporal evolution of interactions been attempted in a high-throughput system. Here, we describe patterns in the outcome of pair-wise co-cultures between two ecologically distinct, yet closely related, strains of the marine cyanobacterium Prochlorococcus and hundreds of heterotrophic marine bacteria. Co-culture with the collection of heterotrophic strains influenced the growth of Prochlorococcus strain MIT9313 much more than that of strain MED4, reflected both in the number of different types of interactions and in the magnitude of the effect of co-culture on various culture parameters. Enhancing interactions, where the presence of heterotrophic bacteria caused Prochlorococcus to grow faster and reach a higher final culture chlorophyll fluorescence, were much more common than antagonistic ones, and for a selected number of cases were shown to be mediated by diffusible compounds. In contrast, for one case at least, temporary inhibition of Prochlorococcus MIT9313 appeared to require close cellular proximity. Bacterial strains whose 16S gene sequences differed by 1-2% tended to have similar effects on MIT9313, suggesting that the patterns of inhibition and enhancement in co-culture observed here are due to phylogenetically cohesive traits of these heterotrophs.

The quinol-fumarate reductase (QFR) respiratory complex of Escherichia coli is a four-subunit integralmembrane complex that catalyzes the final step of anaerobic respiration when fumarate is the terminal electron acceptor. The membrane-soluble redox-active molecule menaquinol (MQH 2 ) transfers electrons to QFR by binding directly to the membrane-spanning region. The crystal structure of QFR contains two quinone species, presumably MQH 2 , bound to the transmembrane-spanning region. The binding sites for the two quinone molecules are termed Q P and Q D , indicating their positions proximal (Q P ) or distal (Q D ) to the site of fumarate reduction in the hydrophilic flavoprotein and iron-sulfur protein subunits. It has not been established whether both of these sites are mechanistically significant. Co-crystallization studies of the E. coli QFR with the known quinol-binding site inhibitors 2-heptyl-4-hydroxyquinoline-N-oxide and 2-[1-(p-chlorophenyl)ethyl] 4,6-dinitrophenol establish that both inhibitors block the binding of MQH 2 at the Q P site. In the structures with the inhibitor bound at Q P , no density is observed at Q D , which suggests that the occupancy of this site can vary and argues against a structurally obligatory role for quinol binding to Q D . A comparison of the Q P site of the E. coli enzyme with quinone-binding sites in other respiratory enzymes shows that an acidic residue is structurally conserved. This acidic residue, Glu-C29, in the E. coli enzyme may act as a proton shuttle from the quinol during enzyme turnover.The Escherichia coli quinol-fumarate reductase (QFR) 1 respiratory complex catalyzes the terminal step of anaerobic respiration when fumarate acts as the terminal electron acceptor (1). During this type of anaerobic respiration, electrons are donated to QFR by menaquinol (MQH 2 ) molecules in the membrane. The electrons are transferred to a covalently-bound flavin adenine nucleotide at the active site through three distinct iron-sulfur clusters and ultimately are used to reduce fumarate to succinate (2, 3). The QFR respiratory complex is composed of four polypeptide chains. Two of these chains, the flavoprotein (FrdA) and the iron protein (FrdB), comprise the soluble domain, which is involved in fumarate reduction, whereas the remaining two subunits (FrdC and FrdD) are membrane-spanning polypeptides involved in the electron transfer with quinones. High sequence identity between the soluble domain of the E. coli QFR and the soluble domain of succinate-quinone oxidoreductase (SQR, complex II) of the aerobic respiratory chain places these two complexes in the same family (4). Consistent with the sequence relationship, both enzymes from E. coli can bidirectionally catalyze the interconversion of succinate and fumarate (5), and each can functionally replace the other to support growth (6, 7). In contrast to the soluble domain, the transmembrane anchor subunits of the complex II family have little sequence identity and exhibit variable polypeptide and cofactor composition. Nevertheless...

To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg(2+) or Mn(2+). The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide gamma-phosphate and its associated Mg(2+) ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.

In this study, we circumvented this problem by taking a heterologous-complementation approach to identify a three-gene operon (the foxEYZ operon) from Rhodobacter sp. strain SW2 that confers enhanced light-dependent Fe(II) oxidation activity when expressed in its genetically tractable relative Rhodobacter capsulatus SB1003. The first gene in this operon, foxE, encodes a c-type cytochrome with no significant similarity to other known proteins. Expression of foxE alone confers significant light-dependent Fe(II) oxidation activity on SB1003, but maximal activity is achieved when foxE is expressed with the two downstream genes foxY and foxZ. In SW2, the foxE and foxY genes are cotranscribed in the presence of Fe(II) and/or hydrogen, with foxZ being transcribed only in the presence of Fe(II). Sequence analysis predicts that foxY encodes a protein containing the redox cofactor pyrroloquinoline quinone and that foxZ encodes a protein with a transport function. Future biochemical studies will permit the localization and function of the Fox proteins in SW2 to be determined.