- Volume 150, Issue 12, 2004

The Weissella paramesenteroides DSMZ 20288T chromosome was analysed by pulsed-field gel electrophoresis, enabling the construction of a physical and genetic map. A total of 21 recognition sites of the restriction enzymes AscI, I-CeuI, NotI and SfiI were mapped on the chromosome, which was found to be circular with an estimated size of 2026 kb. This is believed to constitute the first study into the genomic organization of a strain of this genus, addressing the localization of important chromosomal regions such as oriC and terC. A total of 23 genetic markers were mapped, including eight rrn operons that were precisely assigned in 37 % of the W. paramesenteroides chromosome. The transcription direction of rrn loci was determined and three different rrn clusters were recognized regarding the presence/absence of tRNA genes in ITS regions.

Contact between Helicobacter pylori and gastric epithelial cells results in activation of NF-κB followed by secretion of interleukin (IL)-8. However, host-cell receptor(s) and their ligands involved in H. pylori-related IL-8 production have yet to be fully defined. In this study, the interaction between Toll-like receptors (TLRs), which are host receptors for pathogens involved in the innate immune response, and heat-shock protein (HSP) 60, an immune-potent antigen of H. pylori, was examined during H. pylori-induced IL-8 secretion in vitro. Recombinant H. pylori HSP60 (rHpHSP60) was prepared and added to cultured KATO III human gastric epithelial cells with or without pre-incubation with mouse monoclonal anti-TLR2 or anti-TLR4 antibodies. IL-8 mRNA expression and IL-8 protein release were analysed by Northern blotting and immunoassay. Involvement of NF-κB activation was analysed immunocytochemically by anti-NF-κB p65 antibody and ammonium pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-κB-mediated transcriptional activation. rHpHSP60 induced IL-8 mRNA expression and IL-8 secretion in a dose-dependent manner in KATO III cells. Anti-TLR2 antibody inhibited rHpHSP60-induced IL-8 secretion by 75 %, and anti-TLR4 antibody inhibited it by 30 %. rHpHSP60 induced nuclear translocation of NF-κB p65, which was inhibited by pretreatment with anti-TLR2 antibody. Treatment with PDTC significantly decreased the secretion of IL-8 induced by rHpHSP60. These findings suggest that H. pylori HSP60 activates NF-κB and induces IL-8 production through TLR-triggered pathways in gastric epithelial cells. Thus, it is possible that H. pylori HSP60 and TLR interaction in host cells contributes to the development of gastric inflammation caused by H. pylori infection.

Haemophilus influenzae has an absolute growth requirement for a porphyrin source. This growth requirement can be satisfied in vitro by haem, haemoglobin or the haemoglobin–haptoglobin, haem–haemopexin and haem–albumin complexes. A family of proteins, termed the Hgp proteins, which are essential for utilization of the haemoglobin–haptoglobin complex, has previously been identified. A strain lacking the Hgp proteins also has a residual ability to utilize haemoglobin, indicating that additional moieties contribute to haemoglobin utilization. Using a haemoglobin affinity method an approximately 105 kDa protein was isolated. Mutation of the identified gene in an Hgp null background reduced the ability of the mutant strain to utilize haemoglobin in vitro. The mutation also resulted in a reduced ability to utilize haem, haem–haemopexin, haem–albumin and haemoglobin–haptoglobin, thus identifying a general haem-utilization protein (Hup) in Haemophilus influenzae.

Monoclonal antibodies (MAbs) against Haemophilus parasuis were obtained by the fusion of SP2/0-Ag14 murine myeloma cells and spleen cells from BALB/c mice immunized with a whole-bacterial-cell suspension (WC) of H. parasuis strain SW124 (serotype 4). Two MAbs showing strong reactivity in ELISA were further characterized using SDS-PAGE and Western-blot assays. Different treatments of the WC indicated that MAbs 4D5 and 4G9 identified epitopes of proteinic and polysaccharidic nature, respectively. Electron microscopic examination revealed that, unlike the proteinic epitopes, the lipopolysaccharidic epitopes were exposed on the surface of the cell. Using coagglutination, Western-blot and dot-blot assays it was found that both MAbs recognized common epitopes of all the reference strains and field isolates of H. parasuis. None of the other bacteria tested reacted with the MAbs. These results indicated that both the proteinic and polysaccharidic antigens carried species-specific epitopes. It is suggested that these MAbs may potentially be useful for identification of H. parasuis isolates as well as for developing serological diagnostic tools. MAbs 4D5 and 4G9 were unable to kill H. parasuis in vitro in the presence of complement. However, an enhanced bacterial clearance from blood was observed in mice inoculated with either of the MAbs. Highly significant protection was observed in mice using MAb 4G9. This is believed to be the first report of MAbs capable of identifying common species-specific antigens of H. parasuis and of their implication in protection against challenge infection in mice.

The Yersinia pseudotuberculosis chromosome contains a seven-gene polycistronic unit (the pmrF operon) whose products share extensive homologies with their pmrF counterparts in Salmonella enterica serovar Typhimurium (S. typhimurium), another Gram-negative bacterial enteropathogen. This gene cluster is essential for addition of 4-aminoarabinose to the lipid moiety of LPS, as demonstrated by MALDI-TOF mass spectrometry of lipid A from both wild-type and pmrF-mutated strains. As in S. typhimurium, 4-aminoarabinose substitution of lipid A contributes to in vitro resistance of Y. pseudotuberculosis to the antimicrobial peptide polymyxin B. Whereas pmrF expression in S. typhimurium is mediated by both the PhoP–PhoQ and PmrA–PmrB two-component regulatory systems, it appears to be PmrA–PmrB-independent in Y. pseudotuberculosis, with the response regulator PhoP interacting directly with the pmrF operon promoter region. This result reveals that the ubiquitous PmrA–PmrB regulatory system controls different regulons in distinct bacterial species. In addition, pmrF inactivation in Y. pseudotuberculosis has no effect on bacterial virulence in the mouse, again in contrast to the situation in S. typhimurium. The marked differences in pmrF operon regulation in these two phylogenetically close bacterial species may be related to their dissimilar lifestyles.

The non-haemolytic enterotoxin (Nhe) is one of two three-component enterotoxins responsible for the diarrhoeal food-poisoning syndrome caused by Bacillus cereus. Nhe is composed of NheA, NheB and NheC. The three genes encoding the Nhe components constitute an operon, and the transcriptional start site is located 61 bp upstream of the nheA translational start site. The nhe genes were cloned separately, and expressed in either Bacillus subtilis or Escherichia coli. Separate expression showed that all three components were required for biological activity. In addition, NheA and NheB were purified from B. cereus culture supernatants. As NheC seems to be expressed in only small amounts by B. cereus, NheC was expressed and purified as a histidine-tagged fusion protein. The maximum cytotoxic activity was obtained when the molar ratio between NheA : NheB : His6-NheC was 10 : 10 : 1, and it was shown that NheB was the binding component of the enterotoxin complex.

An important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa is elastase, a secreted thermolysin-like neutral zinc-metalloprotease (TNP). Elastase is synthesized as a larger precursor with an amino-terminal 18 kDa propeptide, and was the first TNP shown to require its propeptide as an intramolecular chaperone (IMC) for activity and secretion. This paper reports the analysis of the elastase propeptide to identify residues conserved among other TNP precursors that may be critical for its IMC function. The prosequences of TNP precursors from both Gram-negative (Vibrio species and Legionella species) and Gram-positive (Bacillus species) bacteria were found to show homology to the elastase propeptide. Two regions of conserved residues were observed: a hydrophilic region (ProM) found in the middle of the elastase propeptide and a more hydrophobic region (ProC) located proximal to the propeptide-processing site. To test whether such conserved motifs were important to function, single residue substitutions at eight conserved amino acids were introduced within the full-length pre-proelastase precursor by site-specific mutagenesis of lasB, the gene encoding elastase. Mutant lasB alleles were expressed from plasmids within a lasB-deleted P. aeruginosa strain, FRD740, and the effects of these propeptide alterations on elastase enzyme activity, processing, stability and accumulation inside and outside of the cell were examined. Within the ProM region, substitution at Arg74 resulted in a dramatic accumulation of proelastase in the cell, suggesting a secretion defect, and a dramatic reduction in supernatant elastolytic activity. Substitution at Asn68 adversely affected the amount of elastase in the culture supernatant, apparently as a result of the reduced stability of the mutated proelastase in the cell. Within the ProC region, mutations at Ile181 and Ala183 abolished the accumulation of a stable elastase molecule in the supernatant. Most mutations produced a phenotype consistent with a defect in protein folding and stability. Overall, the data from this preliminary study show that conserved residues within the elastase propeptide are essential for its function and begin to define the mechanisms of action of IMCs in the TNP family.

The ability of the parasitic flagellate Tritrichomonas foetus to use various iron sources for its physiological requirements was studied. The siderophores ferrioxamine B, ferrichrome, triacetylfusarinine, coprogen, enterobactin and pyoverdine sustained growth of the cells under iron-limited conditions, and siderophore iron was incorporated into the major iron protein of T. foetus, ferredoxin. The kinetics of siderophore uptake by the cells indicated that a non-saturable transport is involved, unlike the uptake of a ferrous salt. Siderophore uptake by the cells did not involve extracellular reductive dissociation of the ferric chelates, although T. foetus cells had some ferrireductase activity on ferric citrate. Fluorescent analogues of siderophores were used to show that the siderophores taken up by the cells were in small intracellular vesicles. The fluorescence emission maximum of pyoverdine in these intracellular vesicles shifted from 460 nm to 530 nm, indicating a very acidic environment. The results suggest that a wide range of chemically unrelated siderophores can be taken up non-specifically and efficiently used by T. foetus; the mechanism involved may be pinocytosis and removal of the iron from the siderophores in acidic intracellular vesicles. Haemin also sustained the growth of T. foetus cells under iron-limited conditions. The use of haemin iron by the cells probably involves haem oxygenase, since traces of biliverdin were found in the medium when haemin was the iron source. The iron uptake and ferrireductase activities of the cells do not seem to be regulated by the amounts of iron and copper in the growth medium.

The genes P1 (MPN141) and ORF6 (MPN142) are essential for the successful colonization of the human respiratory tract by Mycoplasma pneumoniae. Both genes are located in the P1 operon, which consists of three genes. The P1 gene is the second gene in the operon, followed by the ORF6 gene. The P1 gene contains two (RepMP2/3, RepMP4) and the ORF6 gene one (RepMP5) specific repetitive DNA sequence, of which seven to nine similar but not identical copies are dispersed on the genome. Despite this large potential pool for genetic variation, M. pneumoniae isolates from patients contain only one of two distinct combinations of the genes P1 and ORF6. To analyse the functions of the repetitive DNA sequences, two ‘new’ combinations of the genes P1 and ORF6 were constructed, keeping the P1 gene constant but exchanging RepMP5 copies of the ORF6 gene. M. pneumoniae was transformed with these constructs and the transformants were tested for their ability to grow and survive under in vitro conditions and in guinea pigs. The two transformants colonized the respiratory tract of guinea pigs and showed no obvious differences in their growth behaviour compared to M. pneumoniae isolates from patients. The results indicate that the subtype-specific combinations of the repetitive elements in the P1 and ORF6 genes are not essential for the successful adherence of M. pneumoniae to host cells and the colonization of the respiratory tract of guinea pigs.

Several strains of group G streptococci (GGS) form aggregates when grown in vitro. Aggregating strains interact with fibrinogen, and this study reports the isolation of a novel self-associating and fibrinogen-binding protein of GGS, denoted protein FOG. Sequencing of the fog gene revealed structural similarity with M proteins of both GGS and group A streptococci (GAS). Analogous to GAS, GGS were found to multiply in human blood. All strains of GGS express protein G, a protein known to interact with the constant region of immunoglobulin G and albumin. Surprisingly, a clinical isolate expressing protein G, but lacking protein FOG, was killed in human whole blood; however, the addition of intact soluble protein FOG restored the ability of the bacteria to survive and multiply in human blood. This is believed to be the first report of a soluble M-like protein salvaging an M-negative strain from being killed. The antibactericidal property of protein FOG is dependent on its fibrinogen-binding activity. Thus, in plasma, FOG precipitates fibrinogen, and when added to whole blood, protein FOG triggers the formation of visible aggregates comprising fibrinogen and neutrophils that are disabled in their killing of the bacteria. Moreover, the results emphasize the importance of an intact FOG molecule, as presented on the bacterial surface, for full protective effect.

The phenotypes of three different Nitrosomonas europaea strains – wild-type, nitrite reductase (NirK)-deficient and nitric oxide reductase (NorB)-deficient strains – were characterized in chemostat cell cultures, and the effect of nitric oxide (NO) on metabolic activities was evaluated. All strains revealed similar aerobic ammonia oxidation activities, but the growth rates and yields of the knock-out mutants were significantly reduced. Dinitrogen (N2) was the main gaseous product of the wild-type, produced via its denitrification activity. The mutants were unable to reduce nitrite to N2, but excreted more hydroxylamine leading to the formation of almost equal amounts of NO, nitrous oxide (N2O) and N2 by chemical auto-oxidation and chemodenitrification of hydroxylamine. Under anoxic conditions Nsm. europaea wild-type gains energy for growth via nitrogen dioxide (NO2)-dependent ammonia oxidation or hydrogen-dependent denitrification using nitrite as electron acceptor. The mutant strains were restricted to NO and/or N2O as electron acceptor and consequently their growth rates and yields were much lower compared with the wild-type. When cells were transferred from anoxic (denitrification) to oxic conditions, the wild-type strain endogenously produced NO and recovered ammonia oxidation within 8 h. In contrast, the mutant strains remained inactive. For recovery of ammonia oxidation activity the NO concentration had to be adjusted to about 10 p.p.m. in the aeration gas.

During early stationary phase, Bacillus subtilis NAFM5 produces capsular poly(γ-glutamic acid) (γPGA, 2×106 Da), which contains d- and l-glutamate, and then degrades it during late stationary phase. The γ-glutamyltransferase (EC 2.3.2.2; GGT) of this strain successively hydrolysed γPGA from the amino-terminal end, to yield both d- and l-glutamate. This enzyme was specifically synthesized during the stationary phase through transcriptional activation of the corresponding ggt gene by the ComQXPA quorum-sensing system. A ggt knockout mutant degraded γPGA into 1×105 Da fragments, but not any further, indicating that the capsule γPGA is first internally degraded by an endo-type of γPGA hydrolase into 1×105 Da intermediates, then externally into glutamates via GGT. Due to its inability to generate the glutamates from the capsule, the ggt mutant sporulated more frequently than the wild-type strain. The results show that B. subtilis GGT has a powerful exo-γ-glutamyl hydrolase activity that participates in capsule γPGA degradation to supply stationary-phase cells with constituent glutamates.

Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum, produces a mixture of volatile organic compounds (VOCs) in culture and its spectrum of antimicrobial activity is broad. Using the original isolate of M. albus as a selection tool, it has been possible to find other culturally and biochemically unique wild-type isolates of this organism existing as endophytes in a variety of other plant species, including Grevillea pterifolia (fern-leafed grevillea), Kennedia nigriscans (snake vine) and Terminalia prostrata (nanka bakarra) growing in the northern reaches of the Northern Territory of Australia. Interestingly, none of the new isolates had a culture morphology that was identical to the original isolate, nevertheless each possessed hyphal characteristics that resembled that isolate. Furthermore, their ITS-5·8S rDNA sequences were 96–99 % identical to that of M. albus and the isolates were considered M. albus on the basis of the DNA sequence data. However, the VOCs produced by these new isolates greatly differed in quality from the original strain by virtue of the production of naphthalene, naphthalene, 1,1′-oxybis-, and one or more other compounds. In bioassays with a range of test micro-organisms, including fungi and bacteria, each isolate possessed biological activity but the range of activity was great. Artificial mixtures of some of the VOCs mimicked the effects of the VOCs of the fungus. The value of these observations to the biology and practical uses of M. albus in agriculture and other applications is discussed.