- Volume 143, Issue 12, 1997

Newly isolated environmental strains of Comamonas acidovorans and Aureobacterium sp. were found to mineralize sulfoacetate at concentrations up to at least 50 mM. Transient sulfite release was detected during growth on sulfoacetate, with essentially quantitative accumulation of sulfate. Cell-free conversion of sulfoacetate could not be obtained, but resting-cell studies indicated that cleavage of the C-S bonds of both sulfoacetate and sulfoacetaldehyde was induced only when sulfoacetate was the sole carbon and energy source. A sulfite-oxidizing activity was also induced under these conditions. Sulfoacetaldehyde sulfo-lyase activity was demonstrated by in vitro assay and by gel zymography in extracts of cells grown on sulfoacetate as sole carbon source. This activity was not present in acetate-grown cells, or in cells grown on sulfoacetate as sole sulfur source. Results suggest that sulfoacetate mineralization in both isolates may proceed by a novel pathway which involves an initial reduction to sulfoacetaldehyde and subsequent cleavage of the C-S bond to yield sulfite and acetate. The proposed pathway may be of environmental significance in the mineralization of plant sulfolipid.

Paracoccus denitrificans is a heterotrophic organism capable of oxidizing ammonia to nitrite during growth on an organic carbon and energy source. This pathway, termed heterotrophic nitrification, requires the concerted action of an ammonia monooxygenase (AMO) and hydroxylamine oxidase (HAO). The genes required for heterotrophic nitrification have been isolated by introducing a Pa. denitrificans genomic library into Pseudomonas putida and screening for the accumulation of nitrite. In contrast to the situation in chemolithoautotrophic ammonia oxidizers, the genes encoding AMO and HAO are present in single linked copies in the genome of Pa. denitrificans. AMO from Pa. denitrificans expressed in Ps. putida is capable of oxidizing ethene (ethylene) to epoxyethane (ethylene oxide), which is indicative of a relaxed substrate specificity. Further, when expressed in the methylotroph Methylobacterium extorquens AM1, the AMO endows on this organism the ability to grow on ethene and methane. Thus, the Pa. denitrificans AMO is capable of oxidizing methane to methanol, as is the case for the AMO from Nitrosomonas europaea. The heterotrophic nitrification genes are moderately toxic in M. extorquens, more toxic in Ps. putida, and non-toxic in Escherichia coli. Toxicity is due to the activity of the gene products in M. extorquens, and both expression and activity in Ps. putida. This is the first time that the genes encoding an active AMO have been expressed in a heterologous host.

The regulator of fumarate and nitrate reduction (FNR) protein of Escherichia coli is an oxygen-responsive transcription regulator that acts mainly to activate the transcription of genes associated with anaerobic energy generation during periods of oxygen starvation. The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae encodes an FNR homologue, HlyX, which can complement the anaerobic respiratory deficiencies of an fnr mutant. However, FNR and HlyX have distinct but overlapping regulons because during anaerobic incubation, hlyX-expressing E. coli K-12 strains produce an otherwise latent haemolysin. The gene encoding the ‘latent’ haemolysin has been designated hlyE and analysis of the promoter region by DNase I footprinting reveals the presence of an FNR- (HlyX-) binding site. Anaerobic expression of an hlyE::lacZ reporter was 6.5-fold higher in hlyX compared to fnr-expressing cells. Both FNR and HlyX recruited RNA polymerase to the hlyE promoter but formed different ternary complexes. One major transcript (tsp1) initiating at 78.5 bp downstream of the FMR-binding site and four minor transcripts initiating at 73.5 (tsp2), 71.5 (tsp3), 63.5 (tsp4) and 62.5 (tsp5) bp from the FNR site were detected. From the position of the FNR box relative to the transcript starts, hlyE is expressed from a Class I FNR-regulated promoter. Substitution of selected FNR amino acids with the residues found in the equivalent positions in HlyX indicated that Activating Region 1 (AR1) of FNR forms a surface encompassing β9 to β11 and that the AR1 contact at Class I promoters is different to that at Class II promoters, although the same surface is involved. The FNR variant, FNR-A225T, combined the properties of FNR (good activation from Class II promoters) and HlyX (good activation of Class I promoters) and conferred the haemolytic phenotype.

Escherichia colicontains two differentially regulated aconitase genes, acnA and acnB. Two acnA promoters transcribing from start points located 407 bp (P1 acnA) and 50 bp (P2 acnA) upstream of the acnA coding region, and one acnB promoter (P acnB) with a start point 95 bp upstream of the acnB coding region, were identified by primer extension analysis. A 2.8 kb acnA monocistronic transcript was detected by Northern blot hybridization, but only in redox-stressed (methyl-viologen-treated) cultures, and a 2.5 kb acnB monocistronic transcript was detected in exponential- but not stationary-phase cultures. These findings are consistent with previous observations that acnA is specifically subject to SoxRS-mediated activation, whereas acnB encodes the major aconitase that is synthesized earlier in the growth cycle than AcnA. Further studies with acn-lacZ gene fusions and a wider range of transcription regulators indicated that acnA expression is initiated by σ38 from P1 acnA’ and from P2 acnA it is activated directly or indirectly by CRP, FruR, Fur and SoxRS, anc repressed by ArcA and FNR. In contrast, acnB expression is activated by CRP and repressed by ArcA, FruR and Fis from P acnB Comparable studies with fum-lacZ fusions indicated that transcription of fumC, but not of fumA or fumB, is initiated by RNA polymerase containing σ38. It is concluded that AcnB is the major citric acid cycle enzyme, whereas AcnA is an aerobic stationary-phase enzyme that is specifically induced by iron and redox-stress.

The sigB and sigC genes, encoding two alternative sigma factors of the unicellular marine cyanobacterium Synechococcus sp. PCC 7002, were cloned and characterized. Strains in which the sigB and sigC genes were insertionally inactivated were viable under standard laboratory conditions, indicating that SigB and SigC are group 2 sigma factors. Starvation for either nitrogen or carbon caused an increase in sigB mRNA levels. Transcripts for the sigC gene initially increased but then decreased during nitrogen and carbon starvation. The SigC protein could not be identified in cyanobacterial extracts using antisera to Synechococcus sp. PCC 7002 SigA or RpoD from Bacillus subtilis. The ratio of the principal vegetative sigma factor, SigA, to SigB decreased during either nitrogen starvation or carbon starvation, and the levels of SigB also increased in the sigC mutant strain. These results imply that SigB and SigC play roles in modifying transcription in response to changes in carbon and nitrogen availability in this cyanobacterium.

In this study, we describe an unusual illegitimate recombination in the linear-plasmid-encoded outer-surface protein A gene of Borrelia afzelii. A 96 bp DNA segment was deleted from the ospA structural gene of B. afzelii strain R9. The nature of the rearrangement suggested that it arose by a strand slippage mechanism, which was stimulated by a 18-mer palindromic sequence and 5-mer short direct repeats at both termini of the deleted DNA. The deleted sequence could form a complex hairpin structure suggesting that it may have played important roles in pausing of replication and slippaging of the nascent strand across the replication fork. In addition, the mutant strain was isolated from a chronic Lyme disease patient, implying that the variation mechanism may have been used by the borrelial strain to avoid host immune elimination.

To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative σ54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.

Three glycosyltransferases are involved in tylosin biosynthesis in Streptomyces fradiae. The first sugar to be added to the polyketide aglycone (tylactone) is mycaminose and the gene encoding mycaminosyltransferase is orf2 * (tylM2). However, targeted disruption of orf2 * did not lead to the accumulation of tylactone under conditions that normally favour tylosin production; instead, the synthesis of tylactone was virtually abolished. This may, in part, have resulted from a polar effect on the expression of genes downstream of orf2 *. particularly orf4 * (ccr) which encodes crotonyl-CoA reductase, an enzyme that supplies 4-carbon extender units for polyketide metabolism. However, that cannot be the entire explanation, since tylosin production was restored at about 10% of the wild-type level when orf2 * was re-introduced into the disrupted strain. When glycosylated precursors of tylosin were fed to the disrupted strain, they were converted to tylosin, confirming that two of the three glycosyltransferase activities associated with tylosin biosynthesis were still intact. Interestingly, however, tylactone also accumulated under such conditions and, to a much lesser extent, when tylosin was added to similar fermentations. It is concluded that glycosylated macrolides exert a pronounced positive effect on polyketide metabolism in S. fradiae.

During growth and early development of Dictyostelium discoideum, the amoebae exhibit transient pH changes in their cytosol (pHi) and external medium which correlate with the extrusion of H+ from the cell by a plasma membrane pump. Moreover, the changes in pHi have been postulated to influence early prestalk/prespore differentiation during development. To learn more about the role of H+ fluxes in Dictyostelium. we cloned and analysed cDNAs of the gene patB, which appears to encode a P-type H+-ATPase. The patB ORF encodes a protein (termed PAT2) of 1058 amino acids with a calculated molecular mass of 117460 Da. When aligned with other P-type ion-transport ATPases, PAT2 showed the greatest amino acid sequence identity with plasma membrane H+-ATPases of plants and fungi and considerably lower identity with other monovalent cation pumps and with Ca2+ pumps. Northern and Western analyses revealed that patB is expressed at very low levels in cells growing at neutral pH, but it is up-regulated rapidly and dramatically when the cells are shifted to an acidic medium. Immunofluorescence analysis indicated that PAT2 resides on the plasma membrane. When patB was disrupted by homologous recombination, the cells grew and developed normally at neutral and slightly alkaline pHs but they were unable to grow or develop at pH 5.0, and they slowly died. In growth medium at pH 6.8, patB + and patB cells exhibited similar levels of vanadate-sensitive ATPase activity. However, when the cells were shifted to pH 5.0, this activity rapidly increased about twofold in the control cells but not in the mutant cells. Despite the lower ATPase activity in patB cells, they showed relatively normal H+ fluxes and only a slight decrease in pHi when incubated in acidic medium. Together, these results suggest that patB encodes an acid-inducible P-type H+-ATPase which is indispensable for the survival of Dictyostelium cells in moderately acidic external environments.

The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

Two genes, pepQ and orfZ, encoding a prolidase and a prolidase-like protein, respectively, were cloned and characterized from Lactobacillus delbrueckii subsp. bulgaricus. The identity of the pepQ and orfZ genes with the Lactobacillus delbrueckii subsp. lactis prolidase gene (pepQ) was shown to be 98% and 60%, respectively. Both pepQ and orfZ were preceded by a putative promoter region. Northern analysis of pepQ mRNA revealed a 1.1 kb transcript indicating that pepQ forms a monocistronic transcriptional unit. Under the growth conditions used, no evidence was obtained that orfZ was expressed, either by mRNA size determination in Northern analysis or by primer extension analysis. With reverse transcription-PCR, however, the presence of monocistronic orfZ transcripts was established. The orfZ gene could also be overexpressed in E. coli using the vector pKK223-3. The size of the protein synthesized, 41 kDa, confirmed the molecular mass of OrfZ calculated according to DNA sequence analysis. In contrast to PepQ, which showed a substrate specificity characteristic of prolidase enzymes, no enzymic activity for the orfZ-encoded protein was found with the peptide substrates tested. These results indicate that orfZ is a cryptic gene, which is expressed at a very low level under the growth conditions used. It is noteworthy that homologues of the Lb. delbrueckii subsp. bulgaricus orfZ and pepQ genes appeared to be present in both Lb. delbrueckii subsp. lactis and Lactobacillus helveticus.

The spv genes of Salmonella typhimurium and other non-typhoidal Salmonella serovars are essential for efficient systemic infection beyond the intestines in orally inoculated mice as a model for enteric fever. These virulence genes are not significantly expressed by salmonellae during exponential growth in L broth but are induced when the bacteria enter the stationary phase of growth. Using RNase protection analysis to directly measure spvA mRNA from the virulence plasmid of S. typhimurium, we found that spvA was maximally induced in an SpvR- and RpoS-dependent manner during exponential growth in Intracellular Salts Medium, which mimics the intracellular environment of mammalian cells. A cloned spvA-lacZ operon fusion in S. typhimurium was induced intracellularly in peritoneal cells of mice, correlating in vivo intracellular gene expression with intracellular function of the spv genes in infected mice. spvA was also induced intracellularly in vitro within both Henle-407 intestinal epithelial cells and J774.A1 macrophage-like cells when the bacteria were replicating with exponential kinetics. Prevention of invasion of salmonellae with cytochalasin D inhibited spvA induction within tissue culture cells, indicating that salmonellae must be internalized for spvA to be induced. The spvA-lacZ fusion was not induced by salmonellae in extracellular fluid of the peritoneal cavity or in serum. Since induction of the spv genes occurs intracellularly during exponential growth of salmonellae, cessation of growth may not be the most relevant inducing signal for spv gene expression.

Binding of the purified type C 7S (neurotoxin), 12S and 16S botulinum toxins to epithelial cells of ligated small intestine or colon of the guinea pig (in vivo test) and to pre-fixed gastrointestinal tissue sections (in vitro test) was analysed. The 16S toxin bound intensely to the microvilli of epithelial cells of the small intestine in both in vivo and in vitro tests, but did not bind to cells of the stomach or colon. The neurotoxin and 12S toxin did not bind to epithelial cells of the small intestine or to cells of the stomach or colon. Absorption of the toxins was assessed by determining the toxin titre in the sera of guinea pigs 6-8 h after the intra-intestinal administration of the toxins. When the 16S toxin [1 x 105 minimum lethal dose (MLD)] was injected, 200-660 MLD ml-1 was detected in the sera, whereas when the 12S toxin (2 x 105 MLD) or 7S toxin (2 x 105 MLD) was injected, little toxin activity was detected in the sera. Therefore, the haemagglutinin of type C 16S toxin is apparently very important in the binding and absorption of botulinum toxin in the small intestine.

Physico-chemical cell surface properties of 23 coagulase-negative staphylococcal strains, including contact angles, zeta potentials and elemental cell surface composition were measured, together with the adhesion of all strains to hexadecane. The data were employed in a hierarchical cluster analysis, revealing that the 23 strains comprised essentially four different groups. Groups I-III were somewhat similar to each other, but group IV was markedly distinguished from the other strains, predominantly through an elevated acidity of the cell surface. These group distinctions were not related to the presence of a capsule or slime on the strains. Adhesion of the strains to hexadecane depended critically on electrostatic interactions between the hexadecane and the staphylococci, and adhesion only occurred when the electrostatic repulsion between hexadecane and the micro-organisms was less than 500 kT at closest approach. Adhesion of six representative strains from all four groups in a parallel plate flow chamber to silicone rubber, an implant material with similar hydrophobicity to hexadecane, did not show such a critical dependence, nor did it relate with the group distinction Possibly microbial adhesion to substratum surfaces like silicone rubber is more complicated than adhesion to an ideally smooth and homogeneous hexadecane surface in an aqueous solution. Adhesion of all six strains to silicone rubber with an adsorbed conditioning film of plasma proteins was less than that to bare silicone rubber: initial deposition rates dropped from 2000-3000 cm-2 s-1 to 100-300 cm-2 s-1 after adsorption of plasma proteins, while the stationary end-point adhesion decreased from 10 x 106-15 x 106 cm-2 to 1 x 106-5 x 106 cm-2. The adhering staphylococci poorly withstood the passage of an air-bubble through the parallel plate flow chamber regard! of the presence of a conditioning film, indicating a low affinity of these relatively hydrophilic strains for hydrophobic substratum surfaces.