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The type IV bundle-forming pili (BFP) of enteropathogenic Escherichia coli (EPEC) are required for virulence in orally challenged human volunteers and for the localized adherence and autoaggregation in vitro phenotypes. BFP filament biogenesis and function are encoded by the 14-gene bfp operon. The BFP assembly complex, containing a [BfpB-His.sub.6] fusion protein, was chemically cross-linked in situ, and the complex was then purified from BFP-expressing EPEC by a combination of nickel- and BfpB antibody-based affinity chromatography. Characterization of the isolated complex by immunoblotting using BFP protein-specific antibodies showed that at least 10 of the 14 proteins specified by the bfp operon physically interact to form an oligomeric complex. Proteins localized to the outer membrane, inner membrane, and periplasm are within this complex, thus demonstrating that the complex spans the periplasmic space. A combination of immunofluorescence and immuno-gold thin-section transmission electron microscopy studies localized this complex to one pole of the cell.

The Synechocystis sp. strain PCC 6803, which has a T192H mutation in the D2 protein of photosystem II, is an obligate photoheterotroph due to the lack of assembled photosystem II complexes. A secondary mutant, Rg2, has been selected that retains the T192H mutation but is able to grow photoautotrophically. Restoration of photoautotrophic growth in this mutant was caused by early termination at position 294 in the Slr2013 protein. The T192H mutant with truncated Slr2013 forms fully functional photosystem II reaction centers that differ from wild-type reaction centers only by a 30% higher rate of charge recombination between the primary electron acceptor, [Q.sub.A.sup.-], and the donor side and by a reduced stability of the oxidized form of the redox-active Tyr residue, [Y.sub.D], in the D2 protein. This suggests that the T192H mutation itself did not directly affect electron transfer components, but rather affected protein folding and/or stable assembly of photosystem II, and that Slr2013 is involved in the folding of the D2 protein and the assembly of photosystem II. Besides participation in photosystem II assembly, Slr2013 plays a critical role in the cell, because the corresponding gene cannot be deleted completely under conditions in which photosystem II is dispensable. Truncation of Slr2013 by itself does not affect photosynthetic activity of Synechocystis sp. strain PCC 6803. Slr2013 is annotated in CyanoBase as a hypothetical protein and shares a DUF58 family signature with other hypothetical proteins of unknown function. Genes for close homologues of Slr2013 are found in other cyanobacteria (Nostoc punctiforme, Anabaena sp. strain PCC 7120, and Thermosynechococcus elongatus BP-1), and apparent orthologs of this protein are found in Eubacteria and Archaea, but not in eukaryotes. We suggest that Slr2013 regulates functional assembly of photosystem II and has at least one other important function in the cell.

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold ([K.sub.m]= 0.8[+ or -]0.08[micro]M) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 [micro]M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with [K.sub.m] values of 79 and 960 [micro]M, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor ([K.sub.m]=1100[+ or -]140[micro]M; however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on [Mg.sup.2+], and activity was stimulated by potassium, as reflected by a decrease in the [K.sub.m] and an increase in [V.sub.max.] for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.

Highly conserved glycine residues within span I and span II of the phenylalanine and tyrosine transporter PheP were shown to be important for the function of the wild-type protein. Replacement by amino acids with increasing side chain volume led to progressive loss of transport activity. Second-site suppression studies performed with a number of the primary mutants revealed a tight packing arrangement between spans I and acid-polyamine-organocation family, may function as a dimerization motif. Surprisingly, other highly conserved residues, such as Y60 and L41, could be replaced by various residues with no apparent loss of activity.

Activators of [[sigma]sup.54]-RNA polymerase holoenzyme couple ATP hydrolysis to formation of an open promoter complex. [DCtD.sub.[DELTA]1-142], a truncated and constitutively active form of the [sigma]sup.54]-dependent activator DctD from Sinorhizobium meliloti, displayed an altered DNase I footprint at its binding site located upstream of the dctA promoter in the presence of ATP. The altered footprint was not observed for a mutant protein with a substitution at or near the putative arginine finger, a conserved arginine residue thought to contact the nucleotide. These data suggest that structural changes in [DCtD.sub.[DELTA]1-142] during ATP hydrolysis can be detected by alterations in the DNase I footprint of the protein and may be communicated by interactions between bound nucleotide and the arginine finger. In addition, kinetic data for changes in fluorescence energy transfer upon binding of 2'(3')-O-(N-methylanthraniloyl)-ATP (Mant-ATP) to [DCtD.sub.[DELTA]1-142] and DctD suggested that these proteins undergo multiple conformational changes following ATP binding.

The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens. The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins. VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain. Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein. Random mutagenesis identified a unique residue, Met-172, that was required for biological function. Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity. Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.

TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and [sigma]80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1239) activated the transcription of lacZ under the control of the ctx promoter ([P.sub.ctx]::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated [P.sub.ctx]::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

Research reveals structural, organizational, and regulatory diversity in the proline catabolic genes between Pseudomonas aeruginosa and P. putida and the genetic processes involved influence the development of proline catabolism in these bacteria. Data show that the proline utilization system from P. aeruginosa differs from that of P. putida in terms of putA gene.

This article discusses the structure and function of AraC/XylS family of transcription activator proteins and their role in the regulation of virulence factor expression in animal and plant pathogens.

Results reveal that deletion of the FepA globular domain renders the protein insensitive to colicin B and marginally sensitive to colicin D.

Research shows that in mutants harboring a null mutation of phosphatidylglycerophosphate synthase encoding gene pgsA, lipoprotein gene deletion causes unmodified lipoprotein to accumulate in the inner membrane and linking to the peptidoglycan leading to an anmolous anchoring of the inner membrane to peptidoglycan and cell lysis.

The mexR gene encoded product MexR in nalB mutants of Pseudomonas aeruginosa is modified by a single amino acid that compromizes the stability of the protein or its ability to dimerize. However, modification of L95 and R21 amino acid residues confer stability and dimerization capability but compromises DNA binding.

Research reveals that the branched-chain amino acid permease from Rhizobium leguminosarum transports polar amino acids, gamma-aminobutyric acid, hydrophobic-, and neutral amino acids, suggesting that it functions as a global amino acid transporter.

The alpha-galactosidase gene of Bacillus stearothermophilus is transformed into Thermus thermophilus in order to understand the relationship between thermostability and catalytic activity of the alpha-galactosidase at elevated temperatures. Results show that the mutant enzyme exhibits increased temperature optimum, stability, and improved catalytic activity.

Results reveal that the Escherichia coli multidrug transporter MdfA retains its activity of drug resistance and transport in the absence of the entire C-terminal transmembrane domain and the C terrminus as indicated by complementary gene fusion between truncated MdfA-Cat and MdfA-PhoA. The MdfA belongs to the 12-transmembrane-domain family of transporters.

Results show that the mutant sigma(sup)S protein lacking its N-terminal 50 amino acid residues exhibits activity at sigma(sup)S-regulated promoters in vivo, suggesting that the N-terrminal domain is nonessential in Escherichia coli. Furthermore, the induction of sigma(sup)S -specific promoters in stationary phase is regulated by sigma(sup)S protein degradation.

Crystal structure reveals that a 4 degree tilt of the C-terminal domain, encompassing residues 190 through 308, in the apo-TroA zinc-free periplasmic metal-binding protein of Treponema pallidum, renders the protein to exhibit a closed conformation than its counterpart zinc-bound TroA.

Amino acid substitution across the alpha subunit C-terminal domain of Escherichia coli RNA polymerase affects the cyclic AMP receptor protein-dependent transcription at the class I cAMP receptor protein-dependent promoter CC. Amino acid scanning, using alanine, reveals that relative contributions of individual residues within the 265 determinant depends on the promoter sequence.

Results demonstrate that C-terminal truncations of SecY protein translocase, spanning six to seven residues, affect the insertion of the preprotein signal sequence into the membrane. Data indicate that the truncations do not affect SecA binding but impairs the SecA-dependent translocation initiation.

Results show that the two genes srtA and srtB code for sortase enzymes, involved in anchoring the proteins to the cell wall. Data indicate that srtA-derived protein links proteins containing an LPXTG moiff to the cell wall, whereas the srtB gene inactivation affects the surface loalization of proteins.

The DNA sequence analysis, mutagenesis, genetic complementation, and a phage assay reveal that the conjugative transfer region I of the IncHI1 plasmid 27 consists of genes coding for the mating pair formation, DNA transfer replication, and a coupling protein. Mutation in the open reading frames of the transfer region 1 leads to transfer-deficient phenotype.

The nucleic acid polymer translocating functionality of helicases is discussed. The two types of helicases, SuperFamily1 and SuperFamily2, appear to exhibit preference for single- or double-stranded substrates bound to the enzyme through the bases of phosphodiester backbone.

A comparative analysis of the gal-lac gene cluster in Streptococcus salivarius and Streptococcus thermophilus reveals that the Shine-Dalgarno sequences of galT and galE are identical in both, but the ribosome binding site of the latter exhibits two nucleotides difference from that of the former.

Research demonstrates that nucleoid-associated H-NS protein and the global modulator family member Hha protein interact in the absence of DNA in Escherichia coli. Data indicate that the H-NS-binding properties, in relation to alterations in Hha protein, suggest that Hha may be an independent oligomerization domain of H-NS.

The capsule biosynthesis protein CpsB in Streptococcus pneumoniae is a member of the polymerase and histidinol phosphatase family of phosphoesterases. Data suggest that CpsB requires manganese for optimal activity and mutation of the histidine residues inactivates the enzyme.

Results show that the C-terminal region of hemolytic sphingomyelinase of Pseudomonas sp. strain TK4 is important for the hemolytic activity but not essential for the sphingomyelinase activity. In the presence of manganese, the recombinant enzyme hydrolyzes sphingomyelin but not other phospholipids.

The BsuM restriction and modification system of Bacillus subtilis Marburg consists of five open reading frames constituting two operons, the ydiO-ydiP and ydiR-YdiS-ydiA, which express during the logarithmic phase of growth. Data indicate that the ydiO-ydiP mediates modification, while ydiR-ydiS-ydiA that of restriction of the Bsum gene.

Results show that VirB2 protein cleavage of a signal peptide is cyclized to form the T pilin in Agrobacterium tumefaciens. Data indicate that T pilus has a role in the virulence of A. tumefaciens.

Research examines protein-protein interactions between NtrB domains and upstream PII and downstream NtrC components of the nitrogen signal transduction pathway. Data show that interactions between NtrB and NtrC and between NtrB and PII are mediated by histidine phosphotransfer and kinase domains, respectively.

Results reveal that the signal transduction system component ArcB of Haemophilus influenzae although lacks the PAS domain, involved in signal reception of a variety of sensory proteins, still is capable of mediating signal transduction in response to growth conditions.

Results show that the Staphylococcal QacR regulates QacA multidrug transporter and four QacR molecules bind per DNA operator site as dimers.

Results demonstrate that the complete set of Hyp proteins, with the exception of the HypX, mediates the incorporation of the hydrogen-activating [NiFe] site into the regulatory hydrogenase in Ralstonia eutropha. Data indicate that HypF elimination causes a 90% reduction in the regulatory hydrogenase activity.

In shigella base element pairs of ring-proteins MxiM-MxiD and MxiJ-mxiD of the cell envelope form a transmembrane structure, which mediates the assembly of needle complex of the type III secretion pathway. The base element MixM interacts with the major outer-membrane-associated ring-protein MxiD with the former affecting later's stability and structure.

A mutation of uracil to cytosine at position 2609 of 23S ribosomal RNA renders Escherichia coli cells resistant to two ketolides while maintaining susceptibility to other macrolides. Mutational and binding data suggest that the macrolide binding site is located in the nascent peptide exit tunnel.

Results reveal that the outer-membrane lipoprotein BfpB, involved in type IV bundle-forming pili formation, is a multimeric complex whose production and stability are dependent on its interaction with BfpG. Data indicate that BfpB mediates enteropathogenic Escherechia coli protein accumulation in the growth medium.

Results reveal that Bacillus subtilis SpoIVB protein carries a serine peptidase domain and a PDZ domain that are involved in the sporulation process. Data indicate that PDZ domain participates in trans proteolysis and processing of pro-sigma(sup)k and in the signal transduction pathways.

Acute cytotoxicity of epithelial cells and macrophage cell lines is caused by the effector protein ExoU of Pseudomonas aeruginosa. Molecular analysis reveal that N- and C-terminal regions are necessary for toxicity and a middle region of the protein reduces its activity. Data also indicate that ExoU does not oligomerize.

Research describes for the first time biochemical and molecular aspects of purified copper-containing dissimilatory nitrite reductase from Haloarcula marismortui by cloning and sequencing the gene encoding the enzyme. Data indicate differences in some structural, spectroscopic, and catalytic properties of H. marismortui enzyme compared with those from other bacterial species.

Research reveals that the Double-strand DNA repair component RexAB possesses two nuclease motifs, which regulate nuclease activity in Lactococcus lactis. Data show that each of these two motifs participate in the degradation of one DNA strand.

Research describes the details of the colicin Js system in Shigella sonnei 9colicin type 7). Results illustrate the structure of the colicin js coding region on plasmid ColJs, colicin activity gene identification, and Js polypeptide molecular characterization.

The oligomeric PilQ secretin exhibits a donut-like structure with an external ring of 16.5 nanometer dimeter surrounding a 6.5 nanometer diameter central cavity. Data indicate that PilQ is organized as a ring consisting of 12 identical subunits, which serves as a channel for pilus fiber growth in Neisseria meningitidis.

Results show that six genes are involved in the synthesis and assembly of D-galactan I and galactosyltransferases mediating polymerization process. Data indicate that WecA, Wbbo, and WbbM galactosyltransferases are sufficient for the D-galactan I synthesis with Wbbo involved in the polymerization process.

Results reveal that Erwinia chrysanthemi osmoregulated periplasmic glucans exhibit size heterogeneity and structural similarities to Escherichia coli and Pseudomonas syringae osmoregulated periplasmic glucans. Data indicate that the E. chrysanthemi glucan backbones can be substituted with O-acetyl and O-succinyl ester-linked residues.

Results show that the yeast sexual cycle adhesion molecules alpha- and a-agglutinins bind with high affinity with accompanying changes in their secondary structures. At 10 C, the binding kinetics is sigmoidal, suggesting cold sensitivity of sexual agglutination.

Amino acid sequence analysis of purified fimbriae glycoprotein from Streptococcus salivarius reveals that the protein is filamentous in structure and the internal sequence is composed of a repeated motif of two amino acids alternating with two modified residues. Data indicate that the protein is resistant to dissociation.

Research describes cloning, expression, and characterization of the alr1(sp)+ gene and its encoded product alanine racemase ALR1p of Schizosaccharomyces pombe. Data indicate that alanine racemase mediates D-alanine catabolism in S. pombe and enables growth of S. cerevisiae on D-alanine as a sole nitrogen following heterologous expression of the gene.

Results discuss identification of promoter sequences of Actinobacillus pleuropneumoniae by their ability to express promoterless lux genes and of the transcriptional start sites by primer extension analysis. A consensus promoter sequence for A. pleuropneumoniae is presented.

Research shows that cobalamin assimilation mediated 1,2-propanediol degradation by Salmonella enterica is catalyzed by ATP:Cob(I)alamin adenosyltransferase encoded by the pduO gene. Data suggest that the PduO adenosyltransferase is a bifunctional enzyme.

Protection of the trypsin sites in the sensor protein NIFL Q linker by the central domain of the enhancer binding protein NIFA and changes in the trypsin sensitivity of the N-terrminus and Q linker regions of NIFA itself promote the complex formation between the two proteins.

The N-terminal portion of the Shigella virulence factor invasion plasmid antigen B (IpaB) is essential for its expression and the structure of IpaB is preserved by an alpha-helical domain. Data show that 10 residues in the hydrophobic region of the terminus mediate invasion, phagosomal escape, and cytotoxicity.