- Volume 146, Issue 5, 2000

Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His–Era) and glutathione S-transferase (GST)-fusion Era (GST–Era) proteins from Escherichia coli were expressed and purified. It was shown that the GST–Era and His–Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity. However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification. Purified GST–Era protein was shown to be present as a high- and a low-molecular-mass forms. The high-molecular-mass form of GST–Era was associated with RNA and exhibited a much higher GTPase activity. Removal of the RNA associated with GST–Era resulted in a significant reduction in the GTPase activity. The RNA associated with GST–Era was shown to be primarily 16S rRNA. A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA. The native Era protein isolated directly from the cells of a wild-type E. coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a monomer of Era. Furthermore, a C-terminally truncated Era protein, when expressed in E. coli, did not bind RNA. Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein. Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism.

Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil. The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis. An identity of 99·4% was obtained to Mycobacterium gilvum; however, the type strain was unable to utilize morpholine. A maximal growth rate of 0·17 h−1 was observed at a morpholine concentration of 30 mM, 30 °C and pH 7·2. The substrate was tolerated at concentrations up to 100 mM. Besides morpholine, the strain utilized pyrrolidine, piperidine and proposed intermediates in morpholine metabolism such as glycolate, glyoxylate and ethanolamine. Degradation of morpholine, piperidine and pyrrolidine by resting or permeabilized cells was strictly dependent on the presence of oxygen. Addition of the cytochrome-P450-specific inhibitor metyrapone to the growth medium resulted in a significantly decreased growth rate if these cyclic amines were used as a substrate. Carbon monoxide difference spectra of crude extracts from cells grown on these substrates compared to spectra obtained for extracts of succinate-grown cells indicated that cytochrome P450 is specifically expressed during growth on the cyclic amines. These data indicated that a cytochrome-P450-dependent monooxygenase is involved in the degradation of the three cyclic amines.

Biomarkers (respiratory quinones and cellular fatty acids) and denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes were used to characterize the microbial community structure of lab-scale enhanced biological phosphate-removal (EBPR) systems in response to altering sludge phosphorus (P) content. All the data suggest that the microbial community structures of sludge samples with a P content between 8 and 12·3% (sludge dry weight) (i.e. good EBPR activity) were very similar, but differed from those with 2% P content (i.e. no EBPR activity). For all samples analysed, ubiquinones Q-8 and Q-10, menaquinone MK-8(H4), and fatty acids C16:0, C16:1 ω9c and C18:1 ω11c were the major components. The dominance of Q-8, Q-10 and MK-8(H4) suggested that large numbers of organisms belonging to the β and α subclasses of the Proteobacteria and the Actinobacteria from the high G+C Gram-positive bacteria, respectively, were present. DGGE analysis revealed at least 7–9 predominant DNA bands and numerous other fragments in each sample. Five major DGGE fragments from each of the 2% and 12% P-containing sludge samples, respectively, were successfully isolated and sequenced. Phylogenetic analysis of the sequences indicated that both 2% and 12% P-containing sludge samples shared three common phylotypes that were separately affiliated with a novel bacterial group from the γ subclass of the Proteobacteria, two MK-8(H4)-containing actinobacteria previously isolated from the 2% P-containing sludge, and a Caulobacter spp. in the α subclass of the Proteobacteria. The phylogenetic analysis also revealed phylotypes unique to both sludge samples. Changes in sludge P content therefore had an effect on the composition and abundance of the predominant microbial populations, though specific phylotypes could not be unequivocally associated with EBPR.

Cadmium uptake and subcellular compartmentation in the ectomycorrhizal fungus Paxillus involutus were investigated using radiotracer flux analyses. Concentration-dependent Cd2+-uptake kinetics were characterized by a smooth, non-saturating curve that could be dissected into linear and saturable components. The linear-uptake kinetic component was interpreted as representing binding of Cd to apoplastic components, whereas the remaining saturable component was the result of carrier-mediated transport across the plasma membrane. Cell-wall-bound Cd was almost completely removed during desorption from cell-wall preparations. Cd2+ desorption from intact mycelium was found to be a function of time involving three compartments corresponding in series to cell wall (50%), cytoplasm (30%) and vacuole (20%), when mycelia were exposed to a 0·05 μM Cd concentration. At 4 °C, most of the Cd recovered was due to the cell-wall-bound fraction, suggesting that transport across the plasma membrane is a metabolically mediated process. Carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Cd accumulation in P. involutus mycelia by up to 28%, which indicates that transport of Cd2+ was partially dependent on the membrane potential. Cd2+ uptake into symplasm is linked to Ca2+ transport, as revealed by the inhibition of Cd accumulation by the Ca2+ ionophore A23187. The present work demonstrates the ability of the ectomycorrhizal fungus P. involutus to take up and further accumulate Cd in different compartments. Binding of Cd onto cell walls and accumulation of Cd in the vacuolar compartment may be regarded as two essential metal-detoxification mechanisms. These data represent a first step towards the understanding of metal-tolerance mechanisms in mycorrhizal fungi.

An insertion sequence of Mycobacterium fortuitum has been isolated using a trap vector following insertion in and inactivation of the lacZ reporter gene. The trap vector is a temperature-sensitive (ts) Escherichia coli–mycobacterium shuttle plasmid, pCD4, which contains ts oriM, the kanamycin-resistance gene as a selection marker and a lacZ expression cassette. The ts mutation present in pCD4 functions in mycobacteria and enables screening for transposable elements from the mycobacterial genome that disrupt the lacZ gene by screening for white colonies on X-Gal plates in both mycobacterial as well as E. coli hosts. The vector was used to isolate a novel 1·653 kb insertion sequence from M. fortuitum named IS219. IS219 duplicated host DNA at the target site, had inverted repeats at its ends and contained two ORFs on one strand. One of the predicted proteins showed homology to a putative transposase from Acetobacter pasteurianus. IS219 was present in two copies in the genome of M. fortuitum. The trap vector appears to be useful in trapping insertion sequences from different mycobacteria by screening for the disrupted LacZ phenotype.

The analysis of spontaneous bacteriocin-negative mutants has led to the identification and characterization of a new, transpositionally active, insertion sequence of the IS3 family in the lactocin-S-producing Lactobacillus sakei strain L45. The element, which has been designated IS1520, is 1302 bp long with 10 bp perfect inverted repeat ends and generates direct repeats of a trinucleotide of target sequence upon transposition to the lactocin S locus. IS1520 encodes two consecutive, partially overlapping, major ORFs, which are frameshifted in a manner typical of the IS3 family. Despite a low overall DNA sequence similarity, the putative frameshifting region of IS1520 is highly similar to the corresponding region of IS1163, which is a related element previously shown to be active in L. sakei L45.

P ring is a periplasmic substructure of the flagellar basal body and is believed to connect with the peptidoglycan layer in Salmonella. Two flagellar genes, flgA and flgI, are known to be indispensable for P ring formation. The flgI gene encodes the component protein of the P ring. However, the role of the flgA gene product in P ring assembly remained unknown. Here, evidence is presented that FlgA is synthesized as a precursor form and exported via the Sec secretory pathway into the periplasmic space where P ring formation takes place. Overproduction of the FlgI protein led flgA mutants to form flagella with a P ring, suggesting that FlgA plays an auxiliary role in P ring assembly. Far-Western blot analysis revealed that FlgA binds in vitro to both FlgI and FlgA itself. Though a direct FlgI–FlgI interaction in the absence of FlgA could not be demonstrated, an indirect or direct interaction between the FlgI proteins was observed in the presence of FlgA. FlgA alone was very unstable in vivo, but co-expression with FlgI could stabilize FlgA. This suggests the presence of FlgA–FlgI interaction in vivo. On the basis of these results, a hypothesis is proposed that FlgA acts as a periplasmic chaperone, which assists a polymerization reaction of FlgI into the P ring through FlgA–FlgI interaction.

The urease genes of Streptococcus salivarius 57.I are tightly repressed in cells growing at neutral pH. When cells are cultivated at acidic pH values, the urease genes become derepressed and transcription is enhanced when cells are growing under carbohydrate-excess conditions. Previously, the authors proposed that the bacterial sugar:phosphotransferase system (PTS) modulated the DNA-binding activity by phosphorylation of the urease repressor when carbohydrate was limiting. The purpose of this study was to assess whether enzyme I (EI) of the PTS could be involved in modulating urease expression in response to carbohydrate availability. An EI-deficient strain (ptsI18-3) of S. salivarius 57.I was constructed by insertional inactivation of the ptsI gene. The mutant had no measurable PTS activity and lacked EI, as assessed by Western analysis. The mutant grew as well as the wild-type strain on the non-PTS sugar lactose, and grew better than the parent when another non-PTS sugar, galactose, was the sole carbohydrate. The mutant was able to grow with glucose as the sole carbohydrate, but displayed a 24 h lag time and had a generation time some threefold longer than strain 57.I. The mean OD600 attained after 48 h by ptsI18-3 supplied with fructose was 0·16, with no additional growth observed even after 3 d. Urease expression in the wild-type and mutant strains was assessed in continuous chemostat culture. Repression of urease at neutral pH was seen in both strains under all conditions tested. Growth of wild-type cells on limiting concentrations of lactose resulted in very low levels of urease expression compared with growth on PTS sugars. In contrast, under similar conditions, urease expression in ptsI18-3 was restored to levels seen in the parent growing on PTS sugars. Growth under conditions of lactose excess resulted in further derepression of urease, but ptsI18-3 expressed about threefold higher urease activity than 57.I. The results support a role for EI in urease regulation, but also indicate that additional factors may be important in regulating urease gene expression.

Approximately 40–60% of group A streptococcal (GAS) isolates are capable of opacifying sera, due to the expression of the sof (serum opacity factor) gene. The emm (M protein gene) and sof 5′ sequences were obtained from a diverse set of GAS reference strains and clinical isolates, and correlated with M serotyping and anti-opacity-factor testing results. Attempts to amplify sof from strains with M serotypes or emm types historically associated with the opacity-factor-negative phenotype were negative, except for emm12 strains, which were found to contain a highly conserved sof sequence. There was a strong correlation of certain M serotypes with specific emm sequences regardless of strain background, and likewise a strong association of specific anti-opacity-factor (AOF) types to sof gene sequence types. In several examples, M type identity, or partial identity shared between strains with differing emm types, was correlated with short, highly conserved 5′ emm sequences likely to encode M-type-specific epitopes. Additionally, each of three pairs of historically distinct M type reference strains found to share the same 5′ emm sequence, were also found to share M serotype specificity. Based upon sof sequence comparisons between strains of the same and of differing AOF types, an approximately 450 residue domain was determined likely to contain key epitopes required for AOF type specificity. Analysis of two Sof sequences that were not highly homologous, yet shared a common AOF type, further implicated a 107 aa portion of this 450-residue domain in putatively containing AOF-specific epitopes. Taken together, the serological data suggest that AOF-specific epitopes for all Sof proteins may reside within a region corresponding to this 107-residue sequence. The presence of specific, hypervariable emm/sof pairs within multiple isolates appears likely to be a reliable indicator of their overall genetic relatedness, and to be very useful for accurate subtyping of GAS isolates by an approach that has relevance to decades of past M-type-based epidemiological data.

Two closely related pathogens, Bordetella pertussis and Bordetella bronchiseptica, share a number of virulence factors. Filamentous haemagglutinin (FHA) is widely regarded as the dominant adhesin of B. pertussis, and its multiple binding activities have been well characterized. This large protein is produced and secreted at high levels by B. pertussis and significantly lower levels by B. bronchiseptica strains. FHA secretion is mediated by a single outer-membrane accessory protein, FhaC. The genes encoding FHA and FhaC in B. bronchiseptica were characterized by sequencing and functional analyses and are highly similar to those of B. pertussis. The most distinctive feature of B. bronchiseptica FHA is additional repeats in the N-terminal portion of the predicted protein. Interestingly, a point mutation in the fhaB promoter region of the B. bronchiseptica GP1 isolate, relative to other isolates, was found to be detrimental to promoter activity and to FHA production. FhaC and the N-terminal secretion domain of FHA of B. bronchiseptica were fully functional for secretion in B. pertussis. Thus, the different levels of FHA secretion by these Bordetella species might reflect differences in physiology, composition and structure of cell envelope, or differential protein degradation. Characterization of FHA expression and function may provide clues as to the basis of host species tropism, tissue localization and receptor recognition.

A prominent feature of disease induced by Mycoplasma gallisepticum is a lymphoproliferative response in the respiratory tract. Although this is also seen in other mycoplasma infections, including Mycoplasma pneumoniae, the phenotype of the lymphocytes infiltrating the respiratory tract has not been determined. In this study, the numbers and distribution of lymphocytes in the tracheas of chickens infected with a virulent strain of M. gallisepticum were examined. Three groups of chickens were experimentally infected with M. gallisepticum and three unchallenged groups were used as controls. One infected and one control group were culled at 1, 2 and 3 weeks post infection. Tracheas were removed and examined for the presence and number of T cells carrying CD4, CD8, TCRγδ, TCRαβ1 or TCRαβ2 markers. There was no significant difference in the number of CD8+ cells in the upper, middle and lower trachea. High numbers of both CD4+ and CD8+ cells were found with variable numbers of TCRαβ1+ and TCRαβ2+, but no TCRγδ+, cells throughout the time course. The distribution of CD4 cells was dispersed, while the CD8+ cells were clustered in follicular-like arrangements. No difference was detected in the distribution of TCRαβ1+ and TCRαβ2+ cells. The titre of mycoplasma genomes in the trachea decreased significantly from 1 to 2 weeks, while the mucosal thickness of the trachea increased significantly from 1 to 2 weeks then decreased from 2 to 3 weeks, indicating resolution of the lesions following control of infection. This study is the first to examine the phenotypes of T lymphocytes infiltrating the respiratory tract during mycoplasma infections. The findings suggest involvement of specific stimulation of CD8+ cells, particularly in the acute phase of disease.