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Interleukin-10 gene-deficient (II[10.sup.-/-]) mice show a hyper-reaction to normal intestinal bacteria and develop spontaneous colitis similar to that of human Crohn's disease when raised under conventional (but not germ-free) conditions. The lack of ILl0 protein in these mice leads to changes in intestinal metabolic and signalling processes. The first aim of this study was to identify changes in the bacterial community of the caeca at 7 weeks of age (preclinical colitis) and at 12 weeks of age (when clinical signs of colitis are present), and establish if there were any changes that could be associated with the mouse genotype. We have previously shown that dietary n-3 and n-6 polyunsaturated fatty acids (PUFA) have anti-inflammatory effects and affect colonic gene expression profiles in [II.sup.10-/-] mice; therefore, we also aimed to test the effect of the n-3 PUFA eicosapentaenoic acid (EPA) and the n-6 PUFA arachidonic acid (AA) on the bacterial community of caeca in both [II.sup.10-/-] and C57 mice fed these diets. The lower number of caecal bacteria observed before colitis (7 weeks of age) in [II.sup.10-/-] compared to C57 mice suggests differences in the intestinal bacteria that might be associated with the genotype, and this could contribute to the development of colitis in this mouse model. The number and diversity of caecal bacteria increased after the onset of colitis (12 weeks of age). The increase in caecal Escherichia coil numbers in both inflamed [II.sup.10-/-] and healthy C57 mice might be attributed to the dietary PUFA (especially dietary AA), and thus not be a cause of colitis development. A possible protective effect of E. coil mediated by PUFA supplementation and associated changes in the bacterial environment could be a subject for further investigation to define the mode of action of PUFA in colitis. DOI 10.1099/mic.0.041723-0

During the last few years, a substantial body of scientific evidence has accumulated suggesting that certain surface-associated and extracellular components produced by probiotic bacteria could be responsible for some of their mechanisms of action. These bacterial components would be able to directly interact with the host mucosal cells; they include exopolysaccharides, bacteriocins, lipoteichoic acids and surface-associated and extracellular proteins. Extracellular proteins include proteins that are actively transported to the bacterial surroundings through the cytoplasmic membrane, as well as those that are simply shed from the bacterial surface. Compared to the other bacterial components, the interactive ability of extracellular proteins/ peptides has been less extensively studied. In this review, current findings supporting an interaction between extracellular proteins/peptides produced by probiotic bacteria (strains of the genera Bifidobacterium, Lactobacillus and Escherichia) and host mucosal cells are discussed. Research needs and future trends are also considered. DOI 10.1099/mic.0.044057-0

Xanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by CIp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/ dispersal, regulated by the effect of controlled synthesis of DSF on cell--cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-[beta]-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97% identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo- [beta]-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria. DOI 10.1099/mic.0.039420-0

The nucleotide sequence of an open reading frame (corB) downstream of the copper-repressible CorA-encoding gene of the methanotrophic bacterium Methylomicrobium album BG8 was obtained by restriction enzyme digestion and inverse PCR. The amino acid sequence deduced from this gene showed significant sequence similarity to the surface-associated di-haem cytochrome c peroxidase (SACCP) previously isolated from Methylococcus capsulatus (Bath), including both c-type haem-binding motifs. Homology analysis placed this protein, phylogenetically, within the subfamily containing the M. capsulatus SACCP of the bacterial dihaem cytochrome c peroxidase (BCCP) family of proteins. Immunospecific recognition confirmed synthesis of the M. album CorB as a protein non-covalently associated with the outer membrane and exposed to the periplasm, corB expression is regulated by the availability of copper ions during growth and the protein is most abundant in M. album when grown at a low copper-to-biomass ratio, indicating an important physiological role of CorB under these growth conditions. corB was co-transcribed with the gene encoding CorA, constituting a copper-responding operon, which appears to be under the control of a [[sigma].sup.54]-dependent promoter. M. album CorB is the second isolated member of the recently described subfamily of the BCCP family of proteins. So far, these proteins have only been described in methanotrophic bacteria. DOI 10.1099/mic.0.037119-0

In this paper, we describe the regulatory roles of VisN, VisR and Rem in the expression of flagellar, motility and chemotaxis genes in Rhizobium leguminosarum biovar viciae strains VF39SM and 3841. Individual mutations in the genes encoding these proteins resulted in a loss of motility and an absence of flagella, indicating that these regulatory genes are essential for flagellar synthesis and function. Transcriptional experiments involving gusA--gene fusions in wild-type and mutant backgrounds were performed to identify the genes under VisN/R and Rem regulation. Results showed that the chemotaxis and motility genes of R. leguminosarum could be separated into two groups: one group under VisN/R-Rem regulation and another group that is independent of this regulation. VisN and VisR regulate the expression of rem, while Rem positively regulates the expression of flaA, flaB, flaC, flaD, motA, motB, che1 and mcpD. All of these genes except mcpD are located within the main motility and chemotaxis gene cluster of R. leguminosarum. Other chemotaxis and motility genes, which are found outside of the main motility gene cluster (che2 operon, flaH for VF39SM, and flaG) or are plasmid-borne (flaE and mcpC), are not part of the VisN/R-Rem regulatory cascade. In addition, all genes exhibited the same regulation pattern in 3841 and in VF39SM, except flaE and flail, flaE is not regulated by VisN/R-Rem in 3841 but it is repressed by Rem in VF39SM. flail is under VisN/R-Rem regulation in 3841, but not in VF39SM. A kinetics experiment demonstrated that a subset of the flagellar genes is continuously expressed in all growth phases, indicating the importance of continuous motility for R. leguminosarum under free-living conditions. On the other hand, motility is repressed under symbiotic conditions. Nodulation experiments showed that the transcriptional activators VisN and Rem are dramatically downregulated in the nodules, suggesting that the symbiotic downregulation of motility-related genes could be mediated by repressing the expression of VisN/R and Rem. DOI 10.1099/mic.0.035386-0

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes IpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157:H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells. DOI 10.1099/mic.0.034330-0

Many bacteria are motile by means of flagella, semi-rigid helical filaments rotated at the filament's base and energized by proton or sodium-ion gradients. Torque is created between the two major components of the flagellar motor: the rotating switch complex and the cell-wall-associated stators, which are arranged in a dynamic ring-like structure. Being motile provides a survival advantage to many bacteria, and thus the flagellar motor should work optimally under a wide range of environmental conditions. Recent studies have demonstrated that numerous species possess a single flagellar system but have two or more individual stator systems that contribute differentially to flagellar rotation. This review describes recent findings on rotor--stator interactions, on the role of different stators, and on how stator selection could be regulated. An emerging model suggests that bacterial flagellar motors are dynamic and can be tuned by stator swapping in response to different environmental conditions. DOI 10.1099/mic.0.029595-0

Bacillus cereus is a food-berne pathogen and a frequent contaminant of food production plants. The persistence of this pathogen in various environments results from the formation of spores and of biofilms. To investigate the role of the B. cereus flagellar apparatus in biofilm formation, we constructed a non-flagellated mutant and a flagellated but non-motile mutant. Unexpectedly, we found that the presence of flagella decreased the adhesion of the bacterium to g ass surfaces. We hypothesize that this decrease is a consequence of the flagella hindering a direct interaction between the bacterial cell wall and the surface. In contrast, in specific conditions, motility promotes biofilm formation. Our results suggest that motility could influence biofilm formation by three mechanisms. Motility is necessary for the bacteria to reach surfaces suitable for biofilm formation, In static conditions, reaching the air--liquid interface, where the biofilm forms, is a strong requirement, whereas in flow cells bacteria can have access to the bosom glass slide by sedimentation. Therefore, motility is important for biofilm formation in glass tubes and in microtitre plates, but not in flew cells. Motility also promotes recruitment of planktonic cells within the biofilm by allowing motile bacteria to invade the whole biofilm, Finally, motility is involved in the spreading of the biofilm on glass surfaces. DOI 10.1099/mic.0.034827-0

Bacteria growing as surface-adherent biofilms are better able to withstand chemical and physical stresses than their unattached, planktonic counterparts. Using transcriptional profiling and quantitative PCR, we observed a previously uncharacterized gene, yjfO to be upregulated during Escherichia coli MG1655 biofilm growth in a chemostat on serine-limited defined medium. A yjfQ mutant, developed through targeted-insertion mutagenesis, and a yjfQ-complemented strain, were obtained for further characterization. While bacterial surface colonization levels (c.f.u. [cm.sup.-2]) were similar in all three strains, the mutant strain exhibited reduced microcolony formation when observed in flow cells, and greatly enhanced flagellar motility on soft (0.3%) agar. Complementation of yjfO restored microcolony formation and flagellar motility to wild-type levels. Cell surface hydrophobicity and twitching motility were unaffected by the presence or absence of yjfO. In contrast to the parent strain, biofilms from the mutant strain were less able to resist acid and peroxide stresses. yjfO had no significant effect on E. coli biofilm susceptibility to alkali or heat stress. Planktonic cultures from all three strains showed similar responses to these stresses. Regardless of the presence of yjfQ, planktonic E. coli withstood alkali stress better than biofilm populations. Complementation of yjfO restored viability following exposure to peroxide stress, but did not restore acid resistance. Based on its influence on biofilm maturation and stress response, and effects on motility, we propose renaming the uncharacterized gene, yjfO, as bsmA (biofilm stress and motility). DOI 10.1099/mic.0.031468-0

Various nitrate-reducing bacteria produce proteins of the periplasmic nitrate reductase (Nap) system to catalyse electron transport from the membraneous quinol pool to the periptasmic nitrate reductase NapA. The composition of the corresponding nap gene clusters varies but, in addition to napA, genes encoding at least one membrane-bound quinol dehydrogenase module (NapC and/or NapGH) are regularly present. Moreover, some nap loci predict accessory proteins such as the iron-sulfur protein NapF, whose function is poorly understood. Here, the role of NapF in nitrate respiration of the Epsilonproteobacterium Wolinella succinogenes was examined. Immunoblot analysis showed that NapF is located in the membrane fraction in nitrate-grown wild-type cells whereas it was found to be a soluble cytoplasmic protein in a napH deletion mutant. This finding indicates the formation of a membrane-bound NapGHF complex that is likely to catalyse NapH-dependent menaquinol oxidation and electron transport to the iron-sulfur adaptor proteins NapG and NapF, which are located on the periplasmic and cytoplasmic side of the membrane, respectively. The cysteine residues of a CX3CP motif and of the C-terminal tetra-cysteine cluster of NapH were found to be required for interaction with NapF. A napF deletion mutant accumulated the catalytically inactive cytoplasmic NapA precursor, suggesting that electron flow or direct interaction between NapF and NapA facilitated NapA assembly and/or export. On the other hand, NapA maturation and activity was not impaired in the absence of NapH, demonstrating that soluble NapF is functional. Each of the four tetra-cysteine motifs of NapF was modified but only one motif was found to be essential for efficient NapA maturation. It is concluded that the NapGHF complex plays a multifunctional role in menaquinol oxidation, electron transfer to periplasmic NapA and maturation of the cytoplasmic NapA precursor.

Heterogeneity of cells within exponentially growing populations was addressed in a bacterium, the facultative methylotroph Methylobacterium extorquens AM1. A transcriptional fusion between a well-characterized methanol-inducible promoter ([P.sub.mxaF]) and [gfP.sub.uv] was used with flow cytometry to analyse the distribution of gene expression in populations grown on either succinate or methanol, correlated with forward scatter as a measure of cell size. These cell populations were found to consist of three major subpopulations defined by cells that were actively growing and dividing, newly divided, and non-dividing. Through the use of flow cytometry, it was demonstrated that a significant percentage of the total population did not respond to carbon shift. In addition, these experiments demonstrated that a small subset of the total population was significantly brighter than the rest of the population and dominated fluorimetry data. These results were corroborated with a continuous flow-through system and laser scanning microscopy, confirming that subpopulations, not discernible in the population average, dominate population response. These results demonstrate that the combination of flow cytometry and microscopic single-cell analysis can be effectively used to determine the dynamics of subpopulations in population response. In addition, they support the concept that physiological diversity in isogenic populations can poise some proportion of the population to respond appropriately to changing conditions.

A deep-sea manganese-oxidizing bacterium, Brachybacterium sp. strain Mn32, showed high Mn(II) resistance (MIC 55 mM) and Mn(II)-oxidizing/removing abilities. Strain Mn32 removed Mn(II) by two pathways: (1) oxidizing soluble Mn(II) to insoluble biogenic Mn oxides--birnessite ([delta]-Mn[O.sub.2] group) and manganite ([gamma]-MnOOH); (2) the biogenic Mn oxides further adsorb more Mn(II) from the culture. The generated biogenic Mn oxides surround the cell surfaces of strain Mn32 and provide a high capacity to adsorb Zn(II) and Ni(II). Mn(II) oxidation by strain Mn32 was inhibited by both sodium azide and o-phenanthroline, suggesting the involvement of a metalloenzyme which was induced by Mn(II). X-ray diffraction analysis showed that the crystal structures of the biogenic Mn oxides were different from those of commercial pyrolusite ([beta]-Mn[O.sub.2] group) and fresh chemically synthesized vernadite ([delta]-Mn[O.sub.2] group). The biogenic Mn oxides generated by strain Mn32 showed two to three times higher Zn(II) and Ni(II) adsorption abilities than commercial and fresh synthetic Mn[O.sub.2]. The crystal structure and the biogenic Mn[O.sub.2] types may be important factors for the high heavy metal adsorption ability of strain Mn32. This study provides potential applications of a new marine Mn(II)-oxidizing bacterium in heavy metal bioremediation and increases our basic knowledge of microbial manganese oxidation mechanisms.

N-Acylhomoserine lactone (AHL) quorum-sensing (QS) signalling is the best-understood chemical language in proteobacteria. In the last 15 years a large amount of research in several bacterial species has revealed in detail the genetic, molecular and biochemical mechanisms underlying AHL signalling. These studies have revealed the role played by protein pairs of the AHL synthase belonging to the Luxl family and cognate LuxR-family AHL sensor--regulator. Proteobacteria however commonly possess a QS LuxR-family protein for which there is no obvious cognate Luxl synthase; these proteins are found in bacteria which possess a complete AHL QS system(s) as well as in bacteria that do not. Scientists are beginning to address the roles played by these proteins and it is emerging that they could allow bacteria to respond to endogenous and exogenous signals produced by their neighbours. AHL QS research thus far has mainly focused on a cell-density response involving laboratory monoculture studies. Recent findings on the role played by the unpaired LuxR-family proteins highlight the need to address bacterial behaviour and response to signals in mixed communities. Here we review recent progress with respect to these LuxR proteins, which we propose to call LuxR 'solos' since they act on their own without the need for a cognate signal generator. Initial investigations have revealed that LuxR solos have diverse roles in bacterial interspecies and interkingdom communication.

Swarm-cell differentiation in Salmonella enterica serovar Typhimurium (S. typhimurium) results in a biosynthetic mode of growth, despite growing on a rich medium, and cells that have elevated antibiotic resistance. These phenotypes are not a prerequisite for swarm motility. By blocking the switch to anabolic growth using amino acid auxotrophs and screening for the presence of elevated antibiotic resistance in the swarm state, we found that cysteine biosynthesis is crucial for complete swarm-cell differentiation. Mutants were made in each cys biosynthetic operon and all had decreased antibiotic resistance in the swarm state, while swim-cell resistance remained the same as that of wild-type cells. This swarm-state-specific decreased resistance in [DELTA]cys strains could be restored to wild-type levels by the addition of cysteine to swarm medium. Two regulatory mutants, [DELTA]cysB and [DELTA]cysE, failed to swarm unless cysteine was supplemented to the medium. We show that all CysB-responsive operons involved in cysteine biosynthesis are upregulated in the swarm state, even though swarm cells are cultivated on a medium that represses cysteine biosynthesis in the swim state. While swarm medium has sufficient cysteine for growth of S. typhimurium, it does not contain enough for swarm-cell differentiation. We hypothesize that in these cells, the additional cysteine requirement is for use in pathways not directly related to cell growth.

Carbon catabolite repression is an important mechanism allowing efficient carbon source utilization. In the soil bacterium Acinetobacter baylyi, this mechanism has been shown to apply to the aromatic degradative pathways for the substrates protocatechuate, p-hydroxybenzoate and vanillate. In this investigation, transcriptional fusions with the gene for luciferase in the gene clusters for the degradation of benzyl esters, anthranilate, benzoate, hydroxycinnamates and dicarboxylates (are, ant, ben, hca and dca genes) were constructed and established in the chromosome of A. baylyi. The respective strains revealed the presence of strong carbon catabolite repression at the transcriptional level. In all cases, succinate and acetate in combination had the strongest repressing effect, and pyruvate (or lactate in case of the ben and hca genes) allowed the highest expression when these carbon sources were supplied together with the respective inducer. The pattern of repression for the different cosubstrates was similar for all operons investigated and was also observed in the absence of the respective inducing compounds, indicating a mechanism that is independent of the respective specific regulators. Repression by acetate and succinate varied between 88% for the hca genes and 99% for the pca genes.

Using a parallel-plate flow chamber, the hydrodynamic shear forces to prevent bacterial adhesion ([F.sub.prev]) and to detach adhering bacteria ([F.sub.det]) were evaluated for hydrophilic glass, hydrophobic, dimethyldichlorosilane (DDS)-coated glass and six different bacterial strains, in order to test the following three hypotheses. 1. A strong hydrodynamic shear force to prevent adhesion relates to a strong hydrodynamic shear force to detach an adhering organism. 2. A weak hydrodynamic shear force to detach adhering bacteria implies that more bacteria will be stimulated to detach by passing an air-liquid interface (an air bubble) through the flow chamber. 3. DLVO (Derjaguin, Landau, Verwey, Overbeek) interactions determine the characteristic hydrodynamic shear forces to prevent adhesion and to detach adhering micro-organisms as well as the detachment induced by a passing air-liquid interface. [F.sub.prev] varied from 0.03 to 0.70 pN, while [F.sub.det] varied from 0.31 to over 19.64 pN, suggesting that after initial contact, strengthening of the bond occurs. Generally, it was more difficult to detach bacteria from DDS-coated glass than from hydrophilic glass, which was confirmed by air bubble detachment studies. Calculated attractive forces based on the DLVO theory ([F.sub.DLVO]) towards the secondary interaction minimum were higher on glass than on DDS-coated glass. In general, all three hypotheses had to be rejected, showing that it is important to distinguish between forces acting parallel (hydrodynamic shear) and perpendicular (DLVO, air-liquid interface passages) to the substratum surface.

An Aeromonas hydrophila AH-3 miniTn5 mutant unable to produce polar and lateral flagella was isolated, in which the transposon was inserted into a gene whose encoded protein was an orthologue of the Campylobacter jejuni motility accessory factor (Mar) protein. In addition to this gene, several other related genes were found in this cluster that was adjacent to the region 2 genes of the polar flagellum. Mutation of the A. hydrophila AH-3 mar-2, neuB-like, flmD or neuA-like genes resulted in non-motile cells that were unable to swim or swarm due to the absence of both polar and lateral flagella. However, both polar and lateral flagellins were present but were unglycosylated. Although the A. hydrophila AH-3 or Aeromonas caviae Sch3N genes did not hybridize with each other at the nucleotide level, the gene products were able to fully complement the mutations in either bacterium. Furthermore, well-characterized C. jejuni genes involved in flagella glycosylation (Cj1293, -1294 and -1317) were fully able to complement A. hydrophila mutants in the corresponding genes (flmA, flmB and neuB-like). It was concluded that the maf-2, neuB-like, flmD and neuA-like genes are involved in the glycosylation of both the polar and the lateral flagella in Aeromonas strains.

Salmonella thin aggregative fimbriae (Taft; curli) are important in pathogenesis and biofilm formation; however, less is known of their structure and morphogenesis. In the Salmonella agfBAC Taft operon, the transcription and role of agfC have been elusive. In this study, agfBAC transcripts were detected using a sensitive reverse transcriptase technique. Native AgfC was not detected using polyclonal antibodies generated against purified hexahistidine-tagged AgfC; however, in trans expression revealed that AgfC was localized to the periplasm as a mature form. An isogenic [DELTA]agfC mutant displayed an abundance of 20 nm fibres, in addition to native Tafi (5-7 rim), and had an increase in cell surface hydrophobicity. Purified 20 nm fibres were depolymerized under exceptionally stringent conditions to release what proved to be AgfA subunits. This revealed that the 20 nm fibres represented a different form of Tafi. The role of AgfC in Tafi assembly was investigated further using an antibody-capture assay of isogenic Aagf mutants. A soluble antibody-accessible form of AgfA was captured in wild-type (wt), AagfB and AagfF strains, in support of the extracellular nucleation-precipitation pathway of Taft assembly, but not in AagfC or [DELTA]agfE mutants. This indicates that AgfC and AgfE are important for AgfA extracellular assembly, facilitating the synthesis of Tafi.

Salmonella enterica serovar Typhimurium can be internalized by immature dendritic cells (DCs). The interacting host and bacterial molecules initiating this process remain uncharacterized. The objective of this study was to investigate whether specific fimbriae are involved in the early step of binding and uptake of Salmonella by DCs. Type 1 fimbriated S. enterica serovar Typhimurium or recombinant Escherichia coli expressing the type 1 fimbriae showed a significantly greater ability to attach to murine bone-marrow-derived DCs than non-fimbriated bacteria. The FimH adhesin was required for efficient interactions with DCs, since fimbriated fimH mutants were impaired in both binding and internalization. Finally, the internalization involved a FimH-dependent process but did not require sipB, a gene essential for Salmonella-mediated invasion of mammalian epithelial cells. Collectively, these data suggest that the bacterial interaction of DCs through the type 1 fimbrial adhesin FimH is sufficient to target S. enterica serovar Typhimurium for cellular uptake.

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than -7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than -8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80% of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60% of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than -8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis. DOI: 10.1099/mic.0.28881-0

The insect-transmissible strain GII-3 of Spiroplasma citri contains plasmids pSci1-6, five of which (pSci1-5) encode adhesin-like proteins and one (pSci6) encodes protein P32, which has been associated with insect transmissibility. In contrast, S. citri strains ASP-1 and 44, which cannot be transmitted via injection into the leafhopper vector Circulifer haematoceps, lack these proteins and also do not carry plasmids pSci1-6. To further study the apparent relationship between the presence of plasmids and insect transmissibility, plasmids from S. citri GII-3 were introduced into the insect-non-transmissible S. citri strain 44 by electrotransformation using the tetM gene as the selection marker. Tetracycline-resistant transformants were shown to carry one, two or three distinct plasmids. Plasmids pSci1-6 were all detected in the transformants, pSci1 being the most frequently found, alone or together with other plasmids. Selected S. citri 44 transformants having distinct plasmid contents were submitted, separately or in combination, to experimental transmission to periwinkle (Catharanthus roseus) plants via injection into the leafhopper vector. The occurrence of symptomatic plants indicated that, in contrast to S. citri 44, spiroplasmal transformants were transmitted to the host plant, in which they multiplied. Spiroplasma cultures isolated from these infected plants all contained pSci6, leading to the conclusion that, under the experimental conditions used, transformation by pSci6 conferred insect transmissibility to S. citri strain 44. This is believed to be the first report of a phenotypic change associated with transformation of S. citri by natural plasmids.

Most bacterial strains adhere poorly to poly(ethylene oxide) (PEO)-brush coatings, with the exception of a Pseudomonas aeruginosa strain. The aim of this study was to find factors determining whether P. aeruginosa strains do or do not adhere to a PEO-brush coating in a parallel plate flow chamber. On the basis of their adhesion, a distinction could be made between three adhesive and three non-adhesive strains of P. aeruginosa, while bacterial motilities and zeta potentials were comparable for all six strains. However, water contact angles indicated that the adhesive strains were much more hydrophobic than the non-adhesive strains. Furthermore, only adhesive strains released surfactive extracellular substances, which may be engaged in attractive interactions with the PEO chains. Atomic force microscopy showed that the adhesion energy, measured from the retract curves of a bacterial-coated cantilever from a brush coating, was significantly more negative for adhesive strains than for non-adhesive strains (P

Functional interaction between bacterial surface-displayed autoaggregation proteins such as antigen 43(Ag43) of Escherichia coli and motility organelles such as flagella has not previously been described. Here, it has been demonstrated for the first time that Ag43-mediated aggregation can inhibit bacterial motility. Ag43 overexpression produces a dominant aggregation phenotype that overrides motility in the presence of low levels of flagella. In contrast, induction of an increased flagellation state prevents Ag43-mediated aggregation. This phenomenon was observed in naturally occurring subpopulations of E. coli as phase variants expressing and not expressing Ag43 revealed contrasting motility phenotypes. The effects were shown to be part of a general mechanism because other short adhesins capable of mediating autoaggregation (AIDA-I and TibA) also impaired motility. These novel insights into the function of bacterial autoaggregation proteins suggest that a balance between these two systems, i.e. autoaggregation and flagellation, influences motility.

Experiments were done to define the nature of the xylan-derived induction signal for xylanase activity, and evaluate which xylanase genes among the three known ones (xynA, xynB and xynC) are induced by the presence of xylan in Prevotella bryantii [B.sub.1]4. During the later stages of exponential growth on glucose, addition of 0.05 % water-soluble xylan (WS-X) stimulated xylanase formation within 30 min. Xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, arabinose and glucuronic acid all failed to induce the xylanase activity. An acid-ethanol-soluble fraction of WS-X (approximate degree of polymerization 30) enhanced the activity significantly, whereas the acid-ethanol-insoluble fraction had no effect, unless first digested by the cloned P. bryantii XynC xylanase. These results indicate that medium- to large-sized xylo-oligosaccharides are responsible for induction. The transcription of all three known xylanase genes from P. bryantii was upregulated coordinately by addition of WS-X. There have been relatively few investigations into the regulation of xylanase activity in bacteria, and it appears to be unique that medium- to large-sized xylo-oligosaccharides are responsible for induction.

Bacillus anthracis, the aetiological agent of anthrax, synthesizes two surface-layer (S-layer) proteins. S-layers are two-dimensional crystalline arrays that completely cover bacteria. In rich medium, the B. anthracis S-layer consists of Sap during the exponential growth phase. Sap is a modular protein composed of an SLH (S-layer homology)-anchoring domain followed by a putative crystallization domain ([Sap.sub.c]). A projection map of the two-dimensional Sap array has been established on deflated bacteria. In this work, the authors used two approaches to investigate whether [Sap.sub.c] is the crystallization domain. The purified [Sap.sub.c] polypeptide (604 aa) was sufficient to form a crystalline structure, as illustrated by electron microscopy. Consistent with this result, the entire [Sap.sub.c] domain promoted auto-interaction in a bacterial two-hybrid screen developed for the present study. The screen was derived from a system that takes advantage of the Bordetella pertussis cyclase subdomain structure to enable one to identify peptides that interact. A screening strategy was then employed to study [Sap.sub.c] subdomains that mediate interaction. A random library, derived from the [Sap.sub.c] domain, was constructed and screened. The selected polypeptides interacting with the complete [Sap.sub.c] were all larger (155 aa and above) than the mean size of the randomly cloned peptides (approx. 60 residues). This result suggests that, in contrast with observations for other interactions studied with this two-hybrid system, large fragments were required to ensure efficient interaction. It was noteworthy that only one polypeptide, which spanned aa 148-358, was able to interact with less than the complete [Sap.sub.c], in fact, with itself.

Pseudomonas aeruginosa is an opportunistic pathogen which causes a variety of diseases, including respiratory tract infections in patients suffering from cystic fibrosis. Therapeutic treatment of P. aeruginosa infections is still very difficult because the bacteria exhibit high intrinsic resistance against a variety of different antibiotics and, in addition, form stable biofilms, e.g. in the human lung. Several virulence factors are produced by P. aeruginosa, among them the two lectins LecA and LecB, which exert different cytotoxic effects on respiratory epithelial cells and presumably facilitate bacterial adhesion to the airway mucosa. Here, the physiology has been studied of the lectin LecB, which binds specifically to L-fucose. A LecB-deficient P. aeruginosa mutant was shown to be impaired in biofilm formation when compared with the wild-type strain, suggesting an important role for LecB in this process. This result prompted an investigation of the subcellular localization of LecB by cell fractionation and subsequent immunoblotting. The results show that LecB is abundantly present in the bacterial outer-membrane fraction. It is further demonstrated that LecB could be released specifically by treatment of the outer-membrane fraction with p-nitrophenyl [alpha]-L-fucose, whereas treatment with D-galactose had no effect. In contrast, a LecB protein carrying the mutation D104A, which results in a defective sugar-binding site, was no longer detectable in the membrane fraction, suggesting that LecB binds to specific carbohydrate ligands located at the bacterial cell surface. Staining of biofilm cells using fluorescently labelled LecB confirmed the presence of these ligands.

Ruminococcus albus produces fimbria-like structures that are involved with the bacterium's adhesion to cellulose. The subunit protein has been identified in strain 8 (CbpC) and strain 20 (GP25) and both are type IV fimbrial (Pil) proteins. The presence of a pil locus that is organized similarly in both strains is reported here together with the results of an initial examination of a second Pil protein. Downstream of the cbpClgp25 gene (hereafter referred to as pilA1) is a second pilin gene (pilA2). Northern blot analysis of pilA1 and pilA2 transcripts showed that the pilA1 transcript is much more abundant in R. albus 8, and real-time PCR was used to measure pilA1 and pilA2 transcript abundance in R. albus 20 and its adhesion-defective mutant D5. Similar to the findings with R. albus 8, the relative expression of pilA1 in the wild-type strain was 73-fold higher than that of pilA2 following growth with cellobiose, and there were only slight differences between the wild-type and mutant strain in pilA1 and pilA2 transcript abundances, indicating that neither pilA1 nor pilA2 transcription is adversely affected in the mutant strain. Western immunoblots showed that the PilA2 protein is localized primarily to the membrane fraction, and the anti-PilA2 antiserum does not inhibit bacterial adhesion to cellulose. These results suggest that the PilA2 protein plays a role in the synthesis and assembly of type IV fimbriae-like structures by R. albus, but its role is restricted to cell-associated functions, rather than as part of the externalized fimbrial structure.

Paracoccus pantotrophus NKNCYSA utilizes (R)-cysteate (2-amino-3-sulfopropionate) as a sole source of carbon and energy for growth, with either nitrate or molecular oxygen as terminal electron acceptor, and the specific utilization rate of cysteate is about 2 mkat [(kg protein).sup.-1]. The initial degradative reaction is catalysed by an (R)-cysteate : 2-oxoglutarate aminotransferase, which yields 3-sulfopyruvate. The latter was reduced to 3-sulfolactate by an NAD-linked sulfolactate dehydrogenase [3.3 mkat [(kg protein).sup.-1]. The inducible desulfonation reaction was not detected initially in cell extracts. However, a strongly induced protein with subunits of 8 kDa ([alpha]) and 42 kDa ([beta]) was found and purified. The corresponding genes had similarities to those encoding altronate dehydratases, which often require iron for activity. The purified enzyme could then be shown to convert 3-sulfolactate to sulfite and pyruvate and it was termed sulfolactate sulfo-lyase (Suy). A high level of sulfite dehydrogenase was also induced during growth with cysteate, and the organism excreted sulfate. A putative regulator, OrfR, was encoded upstream of suyAB on the reverse strand. Downstream of suyAB was suyZ, which was cotranscribed with suyB. The gene, an allele of tauZ, encoded a putative membrane protein with transmembrane helices (COG2855), and is a candidate to encode the sulfate exporter needed to maintain homeostasis during desulfonation, suyAB-like genes are widespread in sequenced genomes and environmental samples where, in contrast to the current annotation, several presumably encode the desulfonation of 3-sulfolactate, a component of bacterial spores.

A new insertion sequence from Corynebacterium glutamicum ATCC 14999 was isolated and characterized. This IS element, designated IS14999, comprised a 1149 bp nucleotide sequence with 22 bp imperfect terminal inverted repeats. IS14999 carries a single open reading frame of 345 amino acids encoding a putative transposase that appears to have partial homology to IS642, an IS630/Tc1 superfamily element, at the C-terminal region in the amino acid sequence. This indicated that IS14999 belonged to the IS630/Tc1 superfamily, which was first identified in C. glutamicum. IS14999 has a unique distance of 38 amino acid residues between the second and third amino acids in the DDE motif, which is well known as the catalytic centre of transposase. This suggested that IS14999 constituted a new subfamily of the IS630/Tc 1 superfamily. A phylogenetic tree constructed on the basis of amino acid sequences of transposases revealed that this new transposable element was more similar to eukaryotic Tc1/mariner family elements than to prokaryotic IS630 family elements. Added to the fact that IS 14999 was present in only a few C. glutamicum strains, this implies that IS 14999 was probably acquired by a recent lateral transfer event from eukaryotic cells. Analysis of the insertion site in C. glutamicum R revealed that IS14999 appeared to transpose at random and always caused a target duplication of a 5'-TA-3' dinucleotide upon insertion, like the other IS630/Tc1 family elements. These findings indicated that IS14999 could be a powerful tool for genetic manipulation of corynebacteria and related species.

Variable-number tandem repeats (VNTRs) have been shown to be a powerful tool in the determination of evolutionary relationships and population genetics of bacteria. The sequencing of a number of Staphylococcus aureus genomes has allowed the identification of novel VNTR sequences in S. aureus, which are similar to those used in the study of the evolution of Mycobacterium tuberculosis clades. Seven VNTRs, termed staphylococcal interspersed repeat units (SIRUs), distributed around the genome are described, occurring in both unique and multiple sites, and varying in length from 48 to 159 bp. Variations in copy numbers were observed in all loci, within both the sequenced genomes and the UK epidemic methicillin-resistant S. aureus (EMRSA) isolates. Clonally related UK EMRSA isolates were clustered using SIRUs, which provided a greater degree of discrimination than multi-locus sequence typing, indicating that VNTRs may be a more appropriate evolutionary marker for studying transmission events and the geographical spread of S. aureus clades.

The Gram-positive actinobacterium Rhodococcus opacus 1CP is able to utilize several (chloro)aromatic compounds as sole carbon sources, and gene clusters for various catabolic enzymes and pathways have previously been identified. Pulsed-field gel electrophoresis indicates the occurrence of a 740 kb megaplasmid, designated p1CP. Linear topology and the presence of covalently bound proteins were shown by the unchanged electrophoretic mobility after S1 nuclease treatment and by the immobility of the native plasmid during non-denaturing agarose gel electrophoresis, respectively. Sequence comparisons of both termini revealed a perfect 13 bp terminal inverted repeat (TIR) as part of an imperfect 583/587 bp TIR, as well as two copies of the highly conserved centre (GCTXCGC) of a palindromic motif. An initial restriction analysis of p1CP was performed. By means of PCR and hybridization techniques, p1CP was screened for several genes encoding enzymes of (chloro)aromatic degradation. A single maleylacetate reductase gene macA, the clc gene cluster for 4-chloro-/3,5-dichlorocatechol degradation, and the clc2 gene cluster for 3-chlorocatechol degradation were found on p1CP whereas the cat and pca gene clusters for the catechol and the protocatechuate pathways, respectively, were not. Prolonged cultivation of the wild-type strain 1CP under non-selective conditions led to the isolation of the clc-and clc2-deficient mutants 1CP.01 and 1CP.02 harbouring the shortened plasmid variants p1CP.01 (500 kb) and p1CP.02 (400 kb).

Eubacterium pyruvativorans I-[6.sup.T] is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source. This study investigated its metabolic properties and its likely ecological niche. Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate. No single amino acid supported growth, and peptides were required for growth on amino acids. Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation. Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate. L-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated. Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively. Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate. The metabolic strategy of E. pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization. The fermentative niche of E. pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with [C.sub.2] units derived from other substrates to dispose of reducing equivalents.

Many bacteria, including Escherichia coli, have a unique gene that encodes glutamate racemase. This enzyme catalyses the formation of D-glutamate, which is necessary for cell wall peptidoglycan synthesis. However, Bacillus subtilis has two glutamate racemase genes, named racE and yrpC. Since race appears to be indispensable for growth in rich medium, the role of yrpC in D-amino acid synthesis is vague. Experiments with racE- and yrpC-knockout mutants confirmed that race is essential for growth in rich medium but showed that this gene was dispensable for growth in minimal medium, where yrpC executes the anaplerotic role of racE. LacZ fusion assays demonstrated that racE was expressed in both types of media but yrpC was expressed only in minimal medium, which accounted for the absence of yrpC function in rich medium. Neither racE nor yrpC was required for B. subtilis cells to synthesize poly-[gamma]-DL-glutamate ([gamma]-PGA), a capsule polypeptide of D- and L-glutamate linked through a [gamma]-carboxylamide bond. Wild-type cells degraded the capsule during the late stationary phase without accumulating the degradation products, D-glutamate and L-glutamate, in the medium. In contrast, racE or yrpC mutant cells accumulated significant amounts of D- but not L-glutamate. Exogenous D-glutamate utilization was somewhat defective in the mutants and the double mutation of racE and yrpC severely impaired D-amino acid utilization. Thus, both racemase genes appear necessary to complete the catabolism of exogenous D-glutamate generated from [gamma]-PGA.

Environmental signals trigger changes in the bacterial cell surface, including changes in exopolysaccharides and proteinaceous appendages that ultimately favour bacterial persistence and proliferation. Such adaptations are regulated in diverse bacteria by proteins with GGDEF and EAL domains. These proteins are predicted to regulate cell surface adhesiveness by controlling the level of a second messenger, the cyclic dinucleotide c-di-GMP. Genetic evidence suggests that the GGDEF domain acts as a nucleotide cyclase for c-di-GMP synthesis while the EAL domain is a good candidate for the opposing activity, a phosphodiesterase for c-di-GMP degradation.

Activated sludge plants designed to remove phosphorus microbiologically often perform unreliably. One suggestion is that the polyphosphate-accumulating organisms (PAO) are out-competed for substrates by another group of bacteria, the glycogen-accumulating organisms (GAO) in the anaerobic zones of these processes. This study used fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) to analyse the communities from laboratory-scale anaerobic : aerobic sequencing batch reactors. Members of the genus Sphingomonas in the [alpha]-Proteobacteria were present in large numbers in communities with poor phosphorus removal capacity where the biomass had a high glycogen content. Their ability to store poly-[beta]-hydroxyalkanoates anaerobically, but not aerobically, and not accumulate polyphosphate aerobically is consistent with these organisms behaving as GAO there. No evidence was found to support an important role for the [gamma]-Proteobacteria as possible GAO in these communities, although these bacterial populations have been considered in other studies to act as possible competitors for the PAO.

The symbioses between cellulose-degrading flagellates and bacteria are one of the most fascinating phenomena in the complex micro-ecosystem found in the hindgut of lower termites. However, little is known about the identity of the symbionts. One example is the epibiotic bacteria colonizing the surface of hypermastigote protists of the genus Staurojoenina. By using scanning electron microscopy, it was shown that the whole surface of Staurojoenina sp. from the termite Neotermes cubanus is densely covered with long rod-shaped bacteria of uniform size and morphology. PCR amplification of 16S rRNA genes from isolated protozoa and subsequent cloning yielded a uniform collection of clones with virtually identical sequences. Phylogenetic analysis placed them as a new lineage among the Bacteroidales, only distantly related to other uncultivated bacteria in the hindgut of other termites, including an epibiont of the flagellate Mixotricha paradoxa. The closest cultivated relative was Tannerella forsythensis (< 85 % sequence identity). Fluorescence in situ hybridization with a newly designed clone-specific oligonucleotide probe confirmed that these sequences belong to the rod-shaped epibionts of Staurojoenina sp. Transmission electron microscopy confirmed the presence of a Gram-negative cell wall and revealed special attachment sites for the symbionts on the cell envelope of the flagellate host. Based on the isolated phylogenetic position and the specific association with the surface of Staurojoenina sp., we propose to classify this new taxon of Bacteroidales under the provisional name 'Candidatus Vestibaculum illigatum'.

The transcription of the 14 p-gvp genes involved in gas vesicle formation of Halobacterium salinarum PHH1 is driven by the four promoters pA, pD, pF and pO. The regulation of these promoters was investigated in Haloferax volcanii transformants with respect to the endogenous regulatory proteins GvpE and GvpD. Northern analyses demonstrated that the transcription derived from the pA and pD promoters was enhanced by GvpE, whereas the activities of the pF and pO promoters were not affected. Similar results were obtained using promoter fusions with the bgaH reporter gene encoding an enzyme with [beta]-galactosidase activity. The largest amount of specific [beta]-galactosidase activity was determined for pA-bgaH transformants, followed by pF-bgaH and pD-bgaH transformants. The presence of GvpE resulted in a severalfold induction of the pA and pD promoter, whereas the pF promoter was not affected. A lower GvpE-induced pA promoter activity was seen in the presence of GvpD in the pA-bgaH/[DE.sup.ex] transformants, suggesting a function of GvpD in repression. To determine the DNA sequences involved in the GvpE-mediated activation, a 50-nucleotide region of the pA promoter was investigated by 4-nucleotide scanning mutagenesis. Some of these mutations affected the basal transcription, especially mutations in the region of the TATA box and the putative BRE sequence element, and also around position -10. Mutant E, harbouring a sequence with greater identity to the consensus BRE element, showed a significantly enhanced basal promoter activity compared to wild-type. Mutations not affecting basal transcription, but yielding a reduced GvpE-mediated activation, were located immediately upstream of BRE. These results suggested that the transcription activation by GvpE is in close contact with the core transcription machinery.

Two closely related but compatible plasmids of the IncQ-2[alpha] and IncQ-2[beta] groups pTF-FC2 and pTF-F14, were discovered in two acidiphilic chemolithotrophic bacteria. Cross-complementation and cross-regulation experiments by the replication proteins were carried out to discover what changes were necessary when the plasmids evolved to produce two incompatibility groups. The requirement of a pTC-F14 oriV for a RepC DNA-binding protein was plasmid specific, whereas the requirement for the RepA helicase and RepB primase was less specific and could be complemented by the IncQ-2[alpha] plasmid pTC-FC2, and the IncQ-1 [beta] plasmid plE1108. None of the IncQ-1[alpha] plasmid replication proteins could complement the pTC-F14 oriV, and pTC-F14 and RSF1010 were incompatible. This incompatibility was associated with the RepC replication protein and was not due to iteron incompatibility. Replication of pTC-F14 took place from a 5.7 kb transcript that originated upstream of the mobB gene located within the region required for mobilization. A pTC-F14 mobB-lacZ fusion was regulated by the pTC-F14 repB gene product and was plasmid specific, as it was not regulated by the RepB proteins of pTF-FC2 or the IncQ-1[alpha] and IncQ-1[beta] plasmids. Plasmid pTC-F14 appears to have evolved independently functioning iterons and a plasmid-specific RepC-binding protein; it also has a major replication transcript that is independently regulated from that of pTF-FC2. However, the RepA and RepB proteins have the ability to function with either replicon.

Many studies have shown relationships of substratum hydrophobicity, charge or roughness with bacterial adhesion, although bacterial adhesion is governed by interplay of different physico-chemical properties and multiple regression analysis would be more suitable to reveal mechanisms of bacterial adhesion. The formation of a conditioning film of organic compounds adsorbed from seawater affects the properties of substratum surfaces prior to bacterial adhesion, which is a complicating factor in studying the mechanism of bacterial adhesion. In this paper, the impact of conditioning films adsorbed from natural seawater to four polyurethane coatings with different hydrophobicity, elasticity and roughness was studied for three different marine bacterial strains in a multiple linear regression analysis. The water contact angle on hydrophobic coatings decreased on average by 8 degrees and increased on average by the same amount on hydrophilic coatings. These changes were accompanied by increased concentrations of oxygen and nitrogen on the surface as determined by X-ray photoelectron spectroscopy, indicative of adsorption of proteinaceous material. Furthermore, the mean surface roughness increased on average by 4 nm after conditioning film formation. Multiple linear regression analysis revealed that changes in deposition due to conditioning film formation of Marinobacter hydrocarbonoclasticus, Psychrobacter sp. SW5H and Halomonas pacifica in a stagnation-point flow chamber could be explained in a model comprising hydrophobicity and the prevalence of nitrogen-rich components on the surface for the most hydrophobic strain. For the two more hydrophilic strains, deposition was governed by a combination of surface roughness and hydrophobicity. Elasticity was not a factor in bacterial adhesion to conditioning films.

Microcystis sp. has been recognized in recent years as a producer of a high number of secondary metabolites. Among these, peptides that are produced by the non-ribosomal peptide synthetase pathway often show bioactivity or are toxic to humans. The production of particular peptides is specific for individual Microcystis clones, allowing their characterization as chemotypes by analysing the peptidome. The authors studied the in situ diversity of peptides and chemotypes in Microcystis communities from lakes in and around Berlin, Germany, by direct analysis of individual colonies by MALDI-TOF mass spectrometry. From 165 colonies analysed a total of 46 individual peptides could be identified, 21 of which have not been described previously. For six of the new peptides the structures could be elucidated from fragment patterns, while for others only a preliminary classification could be achieved. In most colonies, two to ten individual peptides were detected. In 19 colonies, 16 of which were identified as M. wesenbergii, no peptide metabolites could be detected. The peptide data of 146 colonies were subjected to an ordination (principal components analysis). The principal components were clearly formed by the microcystin variants Mcyst-LR, -RR and -YR, anabaenopeptins B and E/F, a putative microviridin, and a new cyanopeptolin. In the resulting ordination plots most colonies were grouped into five distinct groups, while 40 colonies scattered widely outside these groups. In some cases colonies from different lakes clustered closely, indicating the presence of similar chemotypes in the respective samples. With respect to colony morphology no clear correlation between a chemotype and a morphospecies could be established, but M. aeruginosa, for example, was found to produce predominantly microcystins. In contrast, M. ichthyoblabe colonies were mostly negative for microcystins and instead produced anabaenopeptins. The number of peptides detected in a limited number of samples and the various combinations of peptides in individual Microcystis colonies highlights the immense metabolic potential and diversity of this genus.

Strains deviating in key phenotypic characters, mainly isolated from cases of bovine pneumonia in five European countries, were genotyped in order to examine their genotypic relationship with Pasteurella multocida. Twenty-two strains of Pasteurella avium biovar 2, including variants in indole, xylose and mannitol, 18 strains of Pasteurella canis biovar 2 and variants of this taxon, five strains of P. multocida subsp, septica showing variations in indole and ornithine decarboxylase, nine strains of P. multocida subsp. multocida showing variation in ornithine decarboxylase and mannitol, and type strains of the subspecies of P. multocida were included. Ribotyping was used to examine the relationship of the strains, and 13 types, each containing between one and 20 isolates, were observed. Identical ribotypes were observed in some cases for P. avium biovar 2 and either P. canis biovar 2 or P. multocida subsp, septica. ITS (16S-23S rRNA internal transcribed spacer) fragment-length profiling showed identity of the majority of strains (47 of 52), representing all four taxa, with only five divergent strains. A 16S rRNA sequence comparison of 11 strains representing the main ribotype clusters showed 99*9% similarity to the type strain of P. multocida subsp, multocida, but only 97*4% similarity was obtained to P. canis (biovar 1) and 93*7 % to P. avium (biovar 1). A species-specific PCR test for P. multocida gave a positive result with biovar 2 variants of P. avium and P. canis. DNA-DNA hybridizations between strains of P. multocida, biovar 2 variants of P. avium and P. canis, and P. multocida subsp, septica confirmed similarity at the species level. It is proposed, on the basis of genotypic similarity, that P. multocida be reclassified to include the biovar 2 variants of P. avium and P. canis and that the existence of the biovar 2 variants of P. avium and P. canis is highly questionable. It is concluded that the redefined P. multocida is genotypically homogeneous, although phenotypically diverse lineages exist with respect to ornithine decarboxylase, indole and mannitol, characters that have been regarded as essential for identification to the species level. A formal reclassification of the species is not possible, however, since too few strains have been found to vary in these key characters. Considering the phenotypic diversity of P. multocida, identification will have to depend partly on genotypic methods and the source host also seems important for safe diagnosis.

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is less clear for the thioesterase domains, which seem in certain cases to be dedicated to the hydrolysis/cyclization of their natural substrate, whereas in other cases they are largely permissive.

The replication of the chromosome is among the most essential functions of the bacterial cell and influences many other cellular mechanisms, from gene expression to cell division. Yet the way it impacts on the bacterial chromosome was not fully acknowledged until the availability of complete genomes allowed one to look upon genomes as more than bags of genes. Chromosomal replication includes a set of asymmetric mechanisms, among which are a division in a lagging and a leading strand and a gradient between early and late replicating regions. These differences are the causes of many of the organizational features observed in bacterial genomes, in terms of both gene distribution and sequence composition along the chromosome. When asymmetries or gradients increase in some genomes, e.g. due to a different composition of the DNA polymerase or to a higher growth rate, so do the corresponding biases. As some of the features of the chromosome structure seem to be under strong selection, understanding such biases is important for the understanding of chromosome organization and adaptation. Inversely, understanding chromosome organization may shed further light on questions relating to replication and cell division. Ultimately, the understanding of the interplay between these different elements will allow a better understanding of bacterial genetics and evolution.

In Salmonella enterica, the last step of the synthesis of adenosylcobamide is catalysed by the cobalamin synthase enzyme encoded by the cobs gene of this bacterium. Overexpression of the S. enterica cobS gene in Escherichia coil elicited the accumulation of the phage shock protein PspA, a protein whose expression has been linked to membrane stress. Resolution of inner and outer membranes of S. enterica by isopycnic density ultracentrifugation showed Cobs activity associated with the inner membrane, a result that was confirmed using antibodies against CobS. Computer analysis of the predicted amino acid sequence of CobS suggested it was an integral membrane protein. Results of experiments performed with strains carrying plasmids encoding CobS-alkaline phosphatase or CobS-[beta]-galactosidase protein fusions were consistent with the membrane localization of the CobS protein. Modifications to the predicted model were made based on data obtained from experiments using protein fusions. The function encoded by the cobS orthologue in the methanogenic archaeon Methanobacterium thermoautotrophicum strain [DELTA]H compensated for the lack of CobS during cobalamin synthesis in cobS strains of S. enterica. Cobalamin synthase activity was also detected in a membrane preparation of M. thermoautotrophicum. It was concluded that the assembly of the nucleotide loop of adenosylcobamides in archaea and bacteria is a membrane-associated process. Possible reasons for the association of adenosylcobamide biosynthetic enzymes with the cell membrane are discussed.

Proteus mirabilis has two tandemly arranged flagellin-encoding genes, flaA and flaB. flaA is transcribed from a [[sigma].sup.28] promoter, while flaB is silent, flaA and flaB can undergo reversible rearrangement to produce a set of hybrid genes referred to as flaAB. Flagellins composed of FlaAB protein have a different amino acid sequence and are antigenically distinct from flagellin composed of FlaA, implicating flagellin gene conversion as a putative virulence mechanism for P. mirabilis. The change in amino acid sequence is also hypothesized to alter the filament helix and, hence, affect the motility of FlaAB-expressing strains. To test this hypothesis, the motility of wild-type P. mirabilis was compared with that of a strain, DF1003, locked into the FlaA[B.sup.+] hybrid phase, under conditions of altered ionic strength, pH and viscosity. Cell motion tracking analysis showed that DF1003 has wild-type swimming velocity at physiological conditions, but moves significantly faster and travels further compared to the wild-type at NaCl concentrations greater than 170 mM. DF 1003 is also significantly faster than the wild-type at pH 5.2, 5.8 and 8.2, and at 5 and 10% polyvinylpyrrolidone. Measurements of amplitude and wavelength for isolated flagella subjected to pH 5.8 or 425 mM NaCl showed a loss of helical structure in FlaA flagella compared to FlaAB filaments, a feature that could significantly affect motility under these conditions. These results support a hypothesis that FlaAB flagellin imparts a motile advantage to P. mirabilis in conditions that otherwise may impede bacterial movement. In a broader context, flagellar antigenic variation, commonly thought to serve as means to avoid host defences, may also enhance motility in other bacterial species, thus aiding in the adaptation and survival of the cells.

The ability to utilize dinitrogen as a nitrogen source is an important phenotypic trait in most currently known methanotrophic bacteria (MB). This trait is especially important for acidophilic MB, which inhabit acidic oligotrophic environments, highly depleted in available nitrogen compounds. Phylogenetically, acidophilic MB are most closely related to heterotrophic dinitrogen-fixing bacteria of the genus Beijerinckia. To further explore the phylogenetic linkage between these metabolically different organisms, the sequences of nifH and nifD gene fragments from acidophilic MB of the genera Methylocella and Methylocapsa, and from representatives of Beijerinckia, were determined. For reference, nifH and nifD sequences were also obtained from some type II MB of the alphaproteobacterial Methylosinus/Methylocystis group and from gammaproteobacterial type I MB. The trees constructed for the inferred amino acid sequences of nifH and nifD were highly congruent. The phylogenetic relationships among MB in the NifH and NifD trees also agreed well with the corresponding 16S rRNA-based phylogeny, except for two distinctive features. First, different methods used for phylogenetic analysis grouped the NifH and NifD sequences of strains of the gammaproteobacterial MB Methylococcus capsulatus within a clade mainly characterized by Alphaproteobacteria, including acidophilic MB and type II MB of the Methylosinus/Methylocystis group. From this and other genomic data from Methylococcus capsulatus Bath, it is proposed that an ancient event of lateral gene transfer was responsible for this aberrant branching. Second, the identity values of NifH and NifD sequences between Methylocapsa acidiphila B2 and representatives of Beijerinckia were clearly higher (98.5 and 96.6%, respectively) than would be expected from their 16S rRNA-based relationships. Possibly, these two bacteria originated from a common acidophilic dinitrogen-fixing ancestor, and were subject to similar evolutionary pressure with regard to nitrogen acquisition. This interpretation is corroborated by the observation that, in contrast to most other diazotrophs, M. acidiphila B2 and Beijerinckia spp. are capable of active growth on nitrogen-free media under fully aerobic conditions.

Photorhabdus luminescens is an insect-pathogenic bacterium that forms a symbiosis with specific entomopathogenic nematodes. In this bacterium, a symbiosis-'deficient' phenotypic variant (known as the secondary variant or form II) arises at a low frequency during prolonged incubation. A knock-out mutant was generated of the regulator of a newly identified two-component regulatory system, designated AstR-AstS. Interestingly, this mutation altered the timing of phenotypic switching. Variant cells arose in the mutant strain several days before they did in the wild-type population, suggesting that AstRS is directly or indirectly involved in the genetic mechanism underlying variant cell formation. This mutation also affected motility and antibiotic synthesis. To identify AstRS-regulated genes, a comparative analysis using two-dimensional gel electrophoresis was performed. Seventeen proteins with modified synthesis in stationary phase were identified by mass spectrometry and shown to be involved in electron-transport systems, energy metabolism, iron acquisition and stress responses. The results imply that AstRS is involved in the adaptation of cells to the stationary phase, whilst negatively affecting the competitive advantage of form I cells. The link between AstRS-dependent stationary-phase adaptation and phenotypic variation is discussed.

Two-component signal transduction systems (TCSs) play fundamental roles in bacterial survival and pathogenesis and have been proposed as targets for the development of novel classes of antibiotics. A new coupled assay was developed and applied to analyse the kinetic mechanisms of three new kinds of inhibitors of TCS function. The assay exploits the biochemical properties of the cognate HpkA-DrrA histidine kinase-response regulator pair from Thermotoga maritima and allows multiple turnovers of HpkA, linear formation of phospherylated DrrA, and Michaelis-Menten analysis of inhibitors. The assay was validated in several ways, including confirmation of competitive inhibition by adenosine 5'-[beta],[gamma]-imidotriphosphate (AMP-PNP). The coupled assay, autophosphorylation and chemical cross-linking were used to determine the mechanisms by which several compounds inhibit TCS function. A cyanoacetoacetamide showed non-competitive inhibition with respect to ATP concentration in the coupled assay. The cyanoacetoacetamide also inhibited autophosphorylation of histidine kinases from other bacteria, indicating that the coupled assay could detect general inhibitors of histidine kinase function. Inhibition of HpkA autophosphorylation by this compound was probably caused by aggregation of HpkA, consistent with a previous model for other hydrophobic compounds. In contrast, ethodin was a potent inhibitor of the combined assay, did not inhibit HpkA autophosphorylation, but still led to aggregation of HpkA. These data suggest that ethodin bound to the HpkA kinase and inhibited transfer of the phosphoryl group to DrrA. A peptide corresponding to the phosphorylation site of DrrA appeared to inhibit TCS function by a mechanism similar to that of ethodin, except that autophosphorylation was inhibited at high peptide concentrations. The latter mechanism of inhibition of TCS function is unusual and its analysis demonstrates the utility of these approaches to the kinetic analyses of additional new classes of inhibitors of TCS function.

The biosynthetic gene clusters of the gyrase inhibitors coumermycin [A.sub.1] and clorobiocin contain two different resistance genes (gyr[B.sup.R] and par[Y.sup.R]). Both genes code for B subunits of type II topoisomerases. The authors have now overexpressed and purified the encoded proteins, as well as the corresponding A subunits GyrA and ParX. Expression was carried out in Streptomyces lividans in the form of hexahistidine fusion proteins, allowing purification by nickel affinity chromatography. The complex of GyrA and Gyr[B.sup.R] was found to catalyse ATP-dependent supercoiling of DNA, i.e. to function as a gyrase, whereas the complex of ParX and Par[Y.sup.R] catalysed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This is believed to represent the first topo IV identified in the class of actinobacteria, and the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer.

Anaplasma phagocytophilum is a tick-borne bacterium that is zoonotic in the USA and southern Europe, but although the bacterium is endemic in the UK, no cases of clinical human disease have yet been detected in that country. Potential genomic differences amongst UK and USA isolates were investigated by comparing partial 16S rRNA gene and p44 paralogue sequences amplified by PCR from 10 UK ruminant or tick isolates, with published sequences from USA isolates. No significant clustering among the isolates was resolved by phylogenetic analysis of alignments containing 16S rRNA gene sequences. The structure of predicted proteins encoded by p44 paralogues, amplified from 81 clones obtained from the UK isolates, was similar to that described previously for paralogues from USA isolates. Paralogue sequences did not obviously cluster by country, host species or isolate, but most paralogues were 30-70% similar, making meaningful alignments difficult. Some p44 paralogues from different isolates formed clusters of sequences that were more than 90% similar to one another ('similarity groups'). The paralogues in each cluster were particularly similar in gene regions most likely to code for ligands. In the sample studied, 95% of the similarity groups comprised paralogues from either USA or UK isolates only and occurred with greater frequency amongst paralogues from USA rather than UK isolates. These findings raise the hypothesis that sequences of paralogues in similarity groups may provide an index of adaptation of different 'strains' of A. phagocytophilum to specific reservoir hosts in different geographical locations, and any associations with infectivity for different species including humans.