- Volume 145, Issue 6, 1999

The growth of marine Pseudomonas sp. NCIMB 2021 as continuous cultures in the presence of surfaces of AISI 316 stainless steel allowed the isolation and partial chemical characterization of exopolymers released into the culture medium (free exopolymers), as well as capsular and biofilm exopolymers. Fourier-transform infrared (FTIR) spectroscopy demonstrated the presence of O- and N-acetylation within the carbohydrate moieties and a predominant 310-helical structure of the protein component, highly resistant to hydrogen/deuterium exchange. Differences between the exopolymers were apparent. Relatively less uronic acid residues were detected in the capsular exopolymers compared to either the biofilm or free exopolymers. O- and N-acetylation were greatest in the biofilm exopolymer. SDS-PAGE protein profiles confirmed differences between exopolymers. The secondary structures of proteins determined using FTIR spectroscopy indicated that the capsular exopolymer had reduced helical content and an increased aggregated strand content compared to the biofilm exopolymer. However, the free exopolymer had an increased β-sheet component and a reduced unordered component when compared to the biofilm and capsular exopolymers. These data suggest that exopolymer chemistry varies with cellular mode of growth.

The asgA gene is required for generation of extracellular A signal, which serves as a cell-density signal for fruiting body development in Myxococcus xanthus. The AsgA protein is a histidine protein kinase and consists of a receiver domain that is conserved among response regulators of two-component signal transduction systems, followed by a histidine protein kinase domain that is conserved among sensor proteins of two-component systems. AsgA is thought to function in a signal transduction pathway that leads to expression of genes required for A-signal generation. A genetic suppression analysis of an asgA missense mutation was undertaken in order to identify genes that may provide information regarding the role of AsgA in A-signal generation and fruiting body formation. Twenty-two independent strains containing mutations that suppress asgA473 were isolated by selecting for production of heat-resistant spores under conditions that promote fruiting body development in wild-type cells. Ten of the 22 suppressor strains contained bypass suppressors. All the suppressor strains had direct spore counts at least three to four times greater than the original asgA473 mutant, and three strains had viable counts that exceeded wild-type by more than one order of magnitude. Surprisingly, none of the suppressor strains produced wild-type levels of extracellular A-signal.

Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (P tac ) and the trpA terminator (T trpA ) and then cloned between NotI sites to construct the CAS-Nm (P tac -nptll-T trpA ) and CAS-Gm (P tac /P aacCl -aacCl-T trpA ) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-P tac -nptll-T trpA ) or CAS-GGm (gusA- P tac /P aacCl -aacCl-T trpA ) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing P tac -nptll-T trpA ) and mTn5-Gm (containing gusA-P tac -nptll-T trpA ) and mTn5-GGm (containing gusA-P tac /P aacCl -aacCl-T trpA ) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented. The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other Gram-negative bacteria.

Erysipelothrix rhusiopathiae, the cause of swine erysipelas and human erysipeloid, produces a haemolysin. A recombinant plasmid, pHLY, conferring haemolytic activity on Escherichia coli was isolated from a genomic library of Ery. rhusiopathiae strain Tama-96. This plasmid was stable in RecA- E. coli, but unstable in a RecA+ strain. A spontaneous deletion plasmid, pMini-HLY, also conferring haemolytic activity was derived from pHLY. Two ORFs were detected in pHLY. Analysis of pMini-HLY and other deletion clones established that ORF2 was associated with haemolytic activity. The sequence of ORF1 was highly homologous to those of transposases in the IS30 family. The deletion which generated pMini-HLY was between two short direct repeat (DR) sequences flanking the ORF1 sequence, and there were inverted repeat sequences inside the two DR sequences, suggesting an insertion element. No sequence homology to the deduced amino acid sequence of ORF2 was detected in the databases, but its sequence was characteristic of a surface lipoprotein. Western blot analysis, using antiserum against the 16 kDa protein produced from ORF2, found the protein to be commonly distributed in all Erysipelothrix species.

Biochemical and genetic evidence is presented to demonstrate that the prpE gene of Salmonella typhimurium encodes propionyl-CoA synthetase, an enzyme required for the catabolism of propionate in this bacterium. While prpE mutants used propionate as carbon and energy source, prpE mutants that lacked acetyl-CoA synthetase (encoded by acs) did not, indicating that Acs can compensate for the lack of PrpE in prpE mutants. Cell-free extracts enriched for PrpE catalysed the formation of propionyl-CoA in a propionate-, ATP-, Mg2+- and HS-CoA dependent manner. Acetate substituted for propionate in the reaction at 48% the rate of propionate; butyrate was not a substrate for PrpE. The propionyl-CoA synthetase activity of PrpE was specific for ATP. GTP, ITP, CTP and TTP were not used as substrates by the enzyme. UV-visible spectrophotometry, HPLC and MS data demonstrated that propionyl-CoA was the product of the reaction catalysed by PrpE.

The fliA gene encodes the flagellum-specific sigma factor σ28 in Salmonella typhimurium. The transcription in vivo and in vitro of this gene was analysed and it was found that there are two promoters for the expression of this gene One is a class 2 promoter which is recognized by σ70-RNA polymerase in the presence of the FlhD and FlhC activator proteins. The other is a class 3 promoter which is recognized by σ28-RNA polymerase. Therefore, the fliA operon is under dual positive control from FlhD/FlhC and from FliA itself. The nucleotide sequence downstream of the fliA gene was determined. The sequence contains two ORFs following the fliA gene. On the basis of their sequence homology, it is concluded that these two correspond to the fliZ and fliY genes of Escherichia coli. Northern blot analysis revealed that the fliZ gene is transcribed from the fliA promoters, whereas the fliY gene is transcribed from both the fliA promoters and its own FlhD/FlhC-independent promoter. A fliZ-disruption mutant was constructed by inserting a kanamycin-resistance gene cassette into the fliZ gene on the chromosome. The mutant showed poor motility, and introduction of a fliZ + plasmid into this mutant restored the wild type level of motility. These results suggest that the fliZ gene may be required for expression of maximal motility.

Four genes, dorC, dorD, dorB and dorR of the DMSO respiratory gene cluster of Rhodobacter capsulatus have been identified and sequenced. dorC encodes a pentahaem c-type cytochrome of the NirT class and the derived DorC protein sequence shows highest similarity to TorC from the Escherichia coli trimethylamine-N-oxide (TMAO) respiratory system. Mutagenesis of dorC resulted in the loss of a 46 kDa haem-staining polypeptide from membranes of R. capsulatus. dorD encodes a protein with highest sequence similarity to TorD from the E. coli TMAO respiratory system. DMSO reductase polypeptide (DorA could not be detected in cell-free extracts of a dorD mutant and it is suggested that DorD has a role in stabilizing the DorA apo-protein prior to insertion of the pterin molybdenum cofactor. dorB encodes a protein with highest sequence similarity to NapD of Paracoccus denitrificans. Mutagenesis of dorB reduced the activity of DMSO reductase and led to the accumulation of a large form of the enzyme that is presumed to represent a cytoplasmic precursor polypeptide. It is suggested that DorB has a role in the biogenesis of DMSO reductase prior to its secretion into the periplasm. dorR is transcribed in the opposite direction to dorC. The derived amino acid sequence of DorR indicates that it is a response regulator and mutation of dorR shows that it is essential for expression of the dorCDA operon. Expression of a chromosomal dorA::lacZ fusion was also dependent on the transcriptional regulator Fnr. The intergenic region between dorR and dorC contains four putative binding sites for DorR but no binding site for Fnr was identified.

The DMSO reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor (Moco) and is located in the periplasm. DNA sequence analysis identified four genes involved in the biosynthesis of the Moco (moaA, moaD, moeB and moaC) immediately downstream of the dor (DMSO respiratory) gene cluster. Rhodobacter capsulatus MoaA was expressed in Escherichia coli as a His6-tagged protein. Although, the expressed protein formed inclusion bodies, EPR spectroscopy showed that MoaA contains a [3Fe-4S] cluster. A moaA mutant was constructed and its phenotype indicates that the Moco biosynthetic gene cluster downstream of the dor operon is specific for the biogenesis of DMSO reductase. Two forms of DMSO reductase were purified by immunoaffinity chromatography from the moaA mutant. A mature form of DMSO reductase was located in the periplasm and a precursor form was found in the cytoplasm.

In Candida albicans, three putative histidine kinase genes have been described thus far, including CaSLN1, CaNIK1/COS1 and CaHK1. The encoded proteins for C. albicans, CaSln 1p and CaNik 1p, which are similar to Sln 1p from Saccharomyces cerevisiae and Nik-1 from Neurospora crassa, seem to function in osmoregulation and morphogenesis, respectively. Recently, the isolation of CaHK1, a putative histidine kinase gene from C. albicans has been reported. In addition to the histidine and aspartyl domains located at its C-terminus as previously described, it is shown here that the N-terminal domain of Cahk 1p contains a P-loop motif and a sequence which shows significant homology with the seven C-terminal domains of serine/threonine kinases. The Ser/Thrhomologous domains of Cahk 1p could, in fact, correspond to its sensor sequence. CaHK1 has been mapped to chromosome 2 and gene deletion studies were undertaken to understand its function. Δcahk1 mutants are phenotypically different from any other histidine kinase mutants thus far described either in C. albicans or in any other yeast or filamentous fungus. This study demonstrates that Δcahk1 mutants flocculate extensively in a gene-dosage-dependent manner under conditions which induce germ-tube formation, such as growth in medium 199 (pH 7·5). The flocculation occurs by an interaction along the hyphal surfaces, probably because of the altered expression of one or more hyphal-cell-surface components in the Δcahk1 mutants. These results indicate that CaHK1 could be involved in regulating their expression.

Null mutations were generated in the erg-3 gene of Neurospora crassa by repeat-induced point mutation (RIP). The mutants were viable, lacked ergosterol, were resistant to the steroidal glycoside α-tomatine and were sensitive to the phytoalexins pisatin and biochanin A. RIP was frequently associated with silencing of the hph gene located adjacent to the duplicated erg-3 sequence. The silencing of hph was reversible in the two cases examined and appeared to be due to the spread of cytosine methylation associated with RIP. The erg-3 mutant could be complemented by transformation with recombinant genes that encode proteins chimeric for amino acid sequences from the transmembrane (TM) domain of human lamin B receptor (LBR). This indicates that the LBR TM domain possesses Δ14,15-reductase activity.

Southern blot hybridization analysis revealed that the extrachromosomal DNAs (EC-DNAs) associated with Vaccinium witches’ broom (VAC) and walnut witches’ broom phytoplasmas and various strains of the Italian clover phyllody phytoplasma (ICPh) were highly homologous among themselves but distinct from EC-DNAs of aster yellows related phytoplasmas occurring in the same insect and plant hosts and collected at the same site as the ICPh strains. The EC-DNAs of various strains of the ICPh differed significantly in number and size, more markedly among samples from different host plant species than among samples from the same host plant species. However, experiments on insect-mediated transmission suggested that the size variation is not associated with plant host specificity. Sequence analysis of cloned fragments revealed the presence of highly conserved ORFs (with substantially invariant putative translation products) but also the presence of regions rich in short direct and inverted repeats, which may be the cause of the size variations. The partial sequence of an EC-DNA associated with VAC encoding a putative replication-associated protein indicated their close phylogenetic relationship with geminiviruses.

The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBCScr protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a β-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect palindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that scrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA.

IdiA (iron-deficiency-induced protein A) is a protein expressed at highly elevated levels in Synechococcus sp. strains PCC 6301 and PCC 7942 under Fe-or Mn-limiting growth conditions. Besides being similar to two bacterial Fe-binding proteins, SfuA and FbpA, IdiA shows similarity to two ORFs (slr0513 and slr1295) of Synechocystis sp. PCC 6803. Northern blot analysis detected one transcript of about 1300 nt in RNA extracted from Synechococcus sp. PCC 6301 and PCC 7942 grown under Fe deficiency. The intensity of this transcript was considerably reduced in Fe-sufficient culture. It could be further shown that the regulation of IdiA expression is at the transcriptional level and that transcription and translation of IdiA are closely linked. Primer extension analysis indicated a single transcriptional start site 193 nt upstream of the first presumed translational start codon. Moreover, molecular characterization of the entire 5·8 kb chromosomal HindIII DNA fragment carrying the idiA gene from Synechococcus sp. PCC 6301 led to the identification of six long ORFs in addition to idiA. The two genes adjacent to idiA, and dpsA located 2018 nt downstream of idiA, were insertionally inactivated in Synechococcus sp. PCC 7942 and the corresponding mutants were partially characterized. These experiments provide evidence that the gene products of idiB, located immediately downstream of idiA, and of dpsA are involved in the activation of IdiA expression, since the absence of each of these two gene products prevents the greatly elevated expression of IdiA under nutrient deficiency.

The acetan biosynthetic pathway in Acetobacter xylinum is an ideal model system for engineering novel bacterial polysaccharides. To genetically manipulate this pathway, an Acetobacter strain (CKE5), more susceptible to gene-transfer methodologies, was developed. A new gene, aceP, involved in acetan biosynthesis was identified, sequenced and shown to have homology at the amino acid level with β-D-glucosyl transferases from a number of different organisms. Disruption of aceP in strain CKE5 confirmed the function assigned above and was used to engineer a novel polysaccharide with a pentasaccharide repeat unit.