- Volume 145, Issue 7, 1999

Human enterotoxigenic Escherichia coli (ETEC) produces a type IV pilus termed longus which is encoded on large plasmids in association with colonization factor antigens (CFAs) and enterotoxins. A plasmid-derived 7 kbp BamHI DNA fragment hybridizing with an oligonucleotide probe designed from the amino-terminal amino acid sequence of the denatured 22 kDa structural longus pilin subunit was subcloned and sequenced. DNA sequencing analysis revealed an open reading frame, designated IngA, whose predicted amino acid sequence matched perfectly the N-terminal sequence of LngA obtained by Edman degradation. IngA is the first gene described of the longus gene cluster. Cloned IngA encoded and expressed a prepilin protein of 236 residues with a calculated mass of 25·17 kDa. The prepilin is apparently processed into a mature pilin of 206 residues with a calculated mass of 21·5 kDa. The predicted peptide sequence of IngA showed 78·8 and 37% identity to CFA/III pilin (CofA) of ETEC and the toxin-coregulated pilus (TcpA) of Vibrio cholerae. Peptide sequence homology between IngA and cofA was more prominent towards the amino terminus than within the carboxy region. Like other type IV pilins, LngA contains two cysteine residues towards the carboxy-terminal region. Transmission electron microscopy and immunoblot analysis of ETEC strains expressing either longus or CFA/III detected antigenic differences between native and denatured epitopes of these pili. In addition, differential regulation of pilus expression was identified when ETEC strains were grown in different media. Our data indicate that longus and CFA/III are two distinct but yet highly related type IV pili of ETEC.

Cells of Sinorhizobium meliloti WSM419 showed an adaptive acid-tolerance response when grown at pH 5·8 instead of pH 7·0. Increasing concentrations of calcium in the exposure medium significantly decreased the death rate of WSM419 cells under conditions of acid stress (pH 4·0). The effect of calcium on survival at pH 4·0 however, appears unconnected to exopolysaccharide (EPS), since a strain with a mutation in exoY (Rm0540) responded to calcium in the exposure medium in the same way as its wild-type parent (Rm2011). The concentration of calcium in the growth medium also affected subsequent survival at pH 4·0, and the effect varied with pH. In cells grown at pH 5·8, higher calcium concentrations also markedly increased the rate of synthesis of EPS; this was not seen in cells grown at pH 7·0. 1H NMR spectra for isolated EPS from WSM419 cultures grown at pH 5·8 and pH 7·0 showed that low pH markedly lowered the degree of substitution with acetyl and pyruvyl groups, but not the degree of substitution with succinyl groups; calcium concentration did not affect the pattern of substitution at either pH. For EPS to be involved in the effect of calcium concentration in the growth medium on survival would imply a deleterious effect of the EPS produced at low pH.

The development of a rapid method for measuring intracellular pH (pHi) in single bacterial cells is described. Lactobacillus delbrueckii subsp. bulgaricus and Listeria innocua were used as test organisms. The method is based upon fluorescence microscopy and ratio imaging of cells stained with carboxyfluorescein succinimidyl ester. After staining, the bacteria were immobilized on a membrane filter and transferred to a closed perfusion chamber, allowing control of the cell environment during analysis. The set-up was optimized with regard to the use of neutral-density filters and background subtraction, for determining the excitation ratio 490 nm/435 nm (R490/435) independent of the excitation light intensity, and to reduce photobleaching. This allowed for time-lapse studies with multiple exposures. To study the pHi of Lb. delbrueckii subsp. bulgaricus and L. innocua in response to different extracellular pH (pHex) values, an in vivo calibration curve was constructed in the pHi range 5·0–8·5. Distinct differences between the two cultures were observed. L. innocua maintained a near-neutral pHi almost independently of pHex (5·0–8·0), whereas the pHi of Lb. delbrueckii subsp. bulgaricus decreased with decreasing pHex. In pure and mixed cultures at pHex 5·0, the pHi values of Lb. delbrueckii subsp. bulgaricus and L. innocua were 6·1 ± 0·2 and 7·5 ± 0·2, respectively. This difference in pHi may explain how Lb. delbrueckii subsp. bulgaricus obtains a competitive advantage over L. innocua at low pHex.

A strain of Acinetobacter with potential for bioremediation of heavy metal-contaminated waters was isolated from a wastewater-treatment plant operating an enhanced biological phosphate removal process. NMR and extractive methods showed that polyphosphate accumulated aerobically was degraded under anaerobic conditions both in the presence and absence of cadmium or uranium (0·2–0·5 mM). NMR showed that free phosphate was formed at the expense of polyphosphate, and an extractive technique indicated that this reaction could be stimulated by the presence of UO2 2+ under these conditions. Energy-dispersive X-ray microanalysis demonstrated that only cadmium could enter the cells, and co-localized with intra-cellular granules containing phosphate and other divalent metals. The effects of other environmental parameters on the anaerobic phosphate metabolism were also investigated. Between pH 5·5 and 8·0, phosphate release increased with increasing pH. Between 4°C and 37°C, phosphate release increased with increasing temperature. The presence of nitrate at concentrations of 10 mM and above inhibited anoxic phosphate release, but supplying tungstate in the growth medium prior to anoxic incubation reduced the production of active nitrate reductase and alleviated this effect.

Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K m and V max values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 μM and 2·4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains.

Studies on autotrophic CO2 fixation by the filamentous anoxygenic photosynthetic bacterium Oscillochloris trichoides strain DG-6 demonstrated that, unlike other green bacteria, this organism metabolized CO2 via the reductive pentose phosphate cycle. Both key enzymes of this cycle — ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase — were detected in cell extracts. The main product of ribulose 1,5-bisphosphate-dependent CO2 fixation was 3-phosphoglyceric acid. KCN, which is known to be a competitive inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase, completely in hibited the CO2 assimilation by whole cells as well as by cell extracts of O. trichoides. The 13C/12C carbon isotope fractionation during photoautotrophic growth of O. trichoides was -19·7°/∞, which is close to that obtained for autotrophic organisms that use ribulose-1,5-bisphosphate carboxylase as the primary carboxylation enzyme. Cell extracts of O. trichoides contained all the enzymes of the tricarboxylic acid cycle except 2-oxoglutarate dehydrogenase. No activity of isocitrate lyase, a key enzyme of the glyoxylate shunt, was found in cell extracts of O. trichoides DG-6.

The possibility of intragenic heterogeneity between copies of the long intergenic (16S–23S rDNA) spacer region (LISR) was investigated by specific amplification of this region from 21 Enterococcus faecalis isolates. Three copies of the LISR (rrnA, B and C) were demonstrated by hybridization of the LISR to genomic DNA cleaved with I-Ceul and Smal. When the LISR amplicon was digested with Tsp5091, two known nucleotide substitutions were detected, one 4 nt upstream from the 5′ end of the tRNAala gene (allele rrnB has the Tsp509l site and rrnA and C do not) and the other 22 nt downstream from the 3′ end of the tRNAala gene (rrnC has the Tsp509l site). Sequence differences at these sites were detected at the allelic level (alleles rrnA, B and C) and different combinations of these alleles were designated Tsp Types. Using densitometry to analyse bands from electrophoresis gels, the intra-isolate ratios of the separate alleles (rrnA:rrnB:rrnC) were determined in each Tsp Type: I (0:3:0), II (1:2:0), III (2:0:1), IV (3:0:0), V (2:1:0) and VI (1:1:1). Sequence variation between the three copies of the LISR was confirmed by the detection of at least five other intra-isolate nucleotide substitutions using heteroduplex analysis by conformation-sensitive gel electrophoresis (CSGE) that were not detected by Tsp509I cleavage. Perpendicular denaturing gradient gel electrophoresis was capable of resolving homoduplexes; six to seven out of a possible nine curves were obtained in some isolates. In the isolate where seven curves were obtained one or more further nucleotide substitutions, not detected by Tsp509l cleavage or CSGE, were detected. On the basis of LISR sequence heterogeneity, isolates were categorized into homogeneous (only one allele sequence present) and heterogeneous (two or three allele sequences present). The transition between homogeneous and heterogeneous LISRs may be useful in studying evolutionary mechanisms between E. faecalis isolates.

The ruminococci are an important group of fibrolytic bacteria inhabiting the rumen. Seventeen strains of presumptively identified Ruminococcus were evaluated by a combination of nearly complete and partial 16S rDNA sequence that identified all strains as either Ruminococcus albus or Ruminococcus flavefaciens. All sequences fell into cluster IV of the Clostridia, while other species of ruminococci (eg. Ruminococcus obeum, Ruminococcus gnavus, Ruminococcus lactaris) fall into cluster XlVa of the Clostridia. Ruminococcus cluster IV sequences were used to design a 16S rRNA oligonucleotide probe to assess the relative abundance of target populations in a stable ruminal environment. A stable population (animals fed eight times per day) was established in sheep so that statistically robust comparisons could be made in the absence of variation due to diurnal rumen fluctuations. The steady state populations were sampled six times over a 24 d period and direct microscopic counts (DC), total culturable counts (TCC), and total cellulolytic counts (CEL) were determined. DC and culturable data (TCC and CEL) were compared with relative abundance estimates of Ruminococcus IV and Fibrobacter succinogenes. A combination of the Ruminococcus and F. succinogenes probes accounted for 4·0% of the bacterial population and cellulolytic bacteria (measured by most-probable numbers) were 5·2% of the total culturable count. These data suggest that a major portion of the Ruminococcus and Fibrobacter diversity has been cultured and is represented by available sequences. Steady state populations were measured over several days in three sheep and an estimate of variation in DC, TCC, CEL and 16S-based data were obtained. These variance estimates could be used to determine the theoretical sample sizes required to obtain statistically significant differences under different experimental conditions.