- Volume 143, Issue 11, 1997

A monoclonal antibody (mAb Z3) was produced using BALB/c mice immunized with whole cells of Streptococcus suis serotype 2 reference strain S735. Screening by dot-ELISA showed that mAb Z3, of isotype lgG2b, reacted only with reference strains and field isolates of S. suis serotypes 1, 2 and 1/2. The recognized epitope was demonstrated to be polysaccharide in nature by periodate oxidation, and located in the capsule, since mAb Z3 reacted with purified capsular material by immunoblotting and was able to stabilize the capsule as shown by electron microscopy. Further characterization indicated that mAb Z3 may react specifically with the sialic acid moiety of the capsule, a common constituent of the polysaccharidic capsular material of the three capsular types, since sialidase-treated cells did not react with mAb Z3 in immunoblotting or indirect EILISA. Purified mAb Z3 was shown to significantly increase the rate of phagocytosis of S. suis cells by porcine monocytes and to activate the clearance of bacteria from the circulation in experimentally infected mice. However, mAb Z3 only offered partial protection to mice challenged with a minimal lethal dose. Thus, even though the capsule of S. suis seems to be an important virulence factor, the epitope recognized by mAb Z3 does not appear to be involved in complete protection against infection.

The nucleotide sequence has been determined for a twelve-gene operon of Escherichia coli designated the hyf operon (hyfABCDEFGHIR-focB). The hyf operon is located at 55.8-56.0 min and encodes a putative nine-subunit hydrogenase complex (hydrogenase four or Hyf), a potential formate- and σ54dependent transcriptional activator, HyfR (related to FhlA), and a possible formate transporter, FocB (related to FocA). Five of the nine Hyf-complex subunits are related to subunits of both the E. coli hydrogenase-3 complex (Hyc) and the proton-translocating NADH:quinone oxidoreductases (complex I and Nuo), whereas two Hyf subunits are related solely to NADH:quinone oxidoreductase subunits. The Hyf components include a predicted 523 residue [Ni-Fe] hydrogenase (large subunit) with an N-terminus (residues 1-170) homologous to the 30 kDa or NuoC subunit of complex I. It is proposed that Hyf, in conjunction with formate dehydrogenase H (Fdh-H), forms a hitherto unrecognized respiration-linked proton-translocating formate hydrogenlyase (FHL-2). It is likely that HyfR acts as a formate-dependent regulator of the hyf operon and that FocB provides the Hyf complex with external formate as substrate.

The hypothesis that biodegradable surfactants stimulate the attachment of biodegradation-competent bacteria to surfaces has been re-evaluated using a variant of the surfactant-degrading bacterium Pseudomonas sp. DES1 designated Pseudomonas sp. DES2. This variant was identical to the parental strain in terms of its carbon-utilization patterns and alcohol dehydrogenase and alkylsulfatase complements (enzymes involved in surfactant biodegradation), but differed markedly in its growth characteristics when using sodium dodecyl triethoxysulfate or triethylene glycol dodecyl ether as secondary carbon sources. Pseudomonas sp. DES1 exhibited diauxie in these surfactant-based culture media in contrast to Pseudomonas sp. DES2, which exhibited single-phase growth. Pseudomonas sp. DES2 did not attach to river sediment in a microcosm system when challenged with a dose of either surfactant, although it did biodegrade the substrate. In contrast, Pseudomonas sp. DES1 attached to the river sediment whilst biodegrading the test substrate. It is concluded that the ether-scission system, which is responsible for primary biodegradation of both substrates, is deregulated in Pseudomonas sp. DES2 in contrast to that in Pseudomonas sp. DES1, and that, contrary to a previous hypothesis, biodegradable surfactants do not necessarily stimulate the attachment of biodegradation-competent bacteria during their biodegradation.

Alginate production in Pseudomonas aeruginosa and the associated mucoid phenotype of isolates from cystic fibrosis patients are under the control of the algU mucABCD cluster. This group of genes encodes AlgU, the P. aeruginosa equivalent of the extreme heat shock σ factor σE in Gram-negative bacteria, the AlgU-cognate anti-σ factor MucA, the periplasmic protein MucB and a serine protease homologue, MucD. While mucA, mucB or mucD act as negative regulators of AlgU, the function of mucC is not known. In this study the role of mucC in P. aeruginosa physiology and alginate production has been addressed. Insertional inactivation of mucC in the wild-type P. aeruginosa strain PAO1 did not cause any overt effects on alginate synthesis. However, it affected growth of P. aeruginosa under conditions of combined elevated temperature and increased ionic strength or osmolarity. inactivation of mucC in mucA or mucB mutant backgrounds resulted in a mucoid phenotype when the cells were grown under combined stress conditions of elevated temperature and osmolarity. Each of the stress factors tested separately did not cause comparable effects. The combined stress factors were not sufficient to cause phenotypically appreciable enhancement of alginate production in mucA or mucB mutants unless mucC was also inactivated. These findings support a negative regulatory role of mucC in alginate production by P. aeruginosa, indicate additive effects of muc genes in the regulation of mucoidy in this organism and suggest that multiple stress signals and recognition systems participate in the regulation of algu-dependent functions.

Campylobacter spp. are well recognized as primary pathogens in animals and in people. To isolate and define the genetic regions encoding major surface antigens of Campylobacter hyoilei, genomic DNA of the type strain of the species, RMIT-32A, was cloned into a cosmid vector, pLA2917, in Escherichia coli and the resulting genomic library was screened using antiserum raised to the parent C. hyoilei strain. Six cosmid clones were found to express a series of immunoreactive bands in the 15-25 kDa range. These bands were proteinase K-resistant and were found in the LPS fraction of the cells, suggesting that the recombinant cosmids expressed C. hyoilei lipo-oligosaccharide (LOS) antigen(s). The minimum DNA insert size required for expression of C. hyoilei LOS antigen(s) in E. coli was 11-8 kb. This region was subcloned into the plasmid vector pBR322. The partial sequencing of the 11.8 kb region showed that it contains two ORFs, designated rfbF and rfbP, showing homology with the rfbF gene from Serratia marcescens and the rfbP gene from Salmonella typhimurium. Both genes are involved in LPS synthesis. The region also contained a sequence homologous to the rfaC gene of E. coli and Sal. typhimurium which is involved in core oligosaccharide synthesis.

Although the attenuator located between the ribosomal protein and RNA polymerase gene domains of the Escherichia coli rplKAJLrpoBC operon has a maximum termination efficiency of 80%, the level of termination is diminished with decreasing transcription frequency. In this report, the use of transcriptional fusions to further investigate the mechanism of transcription-frequency-dependent regulation is described. The termination efficiency of two other weak terminators was assayed over a wide range of transcription frequencies programmed by different strength promoters. The results indicated that a decrease in termination efficiency with decreasing transcription frequency is not an inherent property of weak terminators. Deletion of the 165 bp segment located 439-274 bp upstream of the attenuator abrogated the difference in termination efficiency normally seen between high and low levels of transcription. This suggests that a cis-acting site located in this upstream region is necessary for transcription-frequency-dependent modulation of the attenuator's function. However, this site apparently works only in combination with the attenuator, since it did not cause transcription-frequency-dependent modulation when placed upstream of two other weak terminators. Analysis of the readthrough frequencies of single or tandem copies of the attenuator indicated that the transcription complexes which pass through the attenuator have not been converted to termination-resistant complexes in a manner analogous to the N-mediated antitermination system of lambda. Finally, an examination of termination efficiency in three nusA mutants suggested that although NusA increases readthrough at the attenuator it is not directly involved in transcription-frequency-dependent modulation.

Two neighbouring genes, surE and pcm, at 59 min on the Escherichia coli chromosome are both required for stationary-phase survival. Operon fusions of the putative promoter regions in front of surE (P2) or pcm (P3) with the lacZ reporter gene were constructed to study the transcriptional regulation of pcm and surE. Both promoter regions were able to activate -galactosidase activity in a growth-phase-dependent way in either rich or minimal medium. Induction from both promoters reached the highest level in late stationary phase and was independent of the rpoS/katF gene. Spent medium from early as well as late stationary-phase cultures could induce the expression of either promoter even after dialysis or boiling. A high cell density could induce the promoters more rapidly but not to a greater extent. It is proposed that the induction might be correlated with the decline in growth rate of the cells. The induction patterns of either P2 or P3 were very similar, pcm can thus be transcribed from both the P2 and P3 promoters that are regulated in similar ways.

Candida albicans is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients. In this report, the presence of a phospholipase D (PLD) activity in C. albicans, designated CaPLD1, is demonstrated. This is the first description of PLD activity in this organism. CaPLD1 activity was stimulated by inducers of dimorphic transition. Furthermore, transition was stimulated by the addition of exogenous PLD to cells. The addition of 1-propanol to the medium, which resulted in the production of phosphatidylpropanol by CaPLD1 at the expense of the usual product phosphatide acid, delayed the yeast to hypha transition. These results suggest that CaPLD1 may be an important regulator of dimorphic transition in C. albicans.

A search for Candida albicans mutants defective in filamentous growth led to the isolation of a mutant strain with an insertion mutation in the SEC14 gene. SEC14 encodes the phosphatidylinositol/phosphatidylcholine transfer protein, an essential protein in the yeast Saccharomyces cerevisiae. In the dimorphic yeast Yarrowia lipolytica, SEC14 is needed for growth only in the hyphal form and is not required for growth in the yeast form. However, unlike Y. lipolytica SEC14, C. albicans SEC14 is probably essential for growth. Northern blot analysis and PCR amplification of transcripts produced from the SEC14 gene demonstrated that two transcripts differing at their 3′ ends were produced. The two transcripts may regulate the activity of SEC14 so that Sec14p can perform two functions in C. albicans. One function may be an essential function analogous to the function of Sec14p in S. cerevisiae and the second function may be important during filamentous growth, analogous to the function of Sec14p in Y. lipolytica.

The gene for thermostable 1,3-β-glucosidase BgIB was cloned from the chromosome of Thermotoga neapolitana and its primary sequence was determined. The purified recombinant β-glucosidase B had a monomer molecular mass of 81 kDa in accordance with the amino acid sequence predicted from the nucleotide sequence of clone pTT51. It was a member of glycosylhydrolase family 3 and belonged to enzyme class EC 3.2.1.21. β-Glucosidase B had a specific activity of 255 U mg-1on 4-nitrophenyl(PNP)-β-glucoside at the optima of pH (5.5) and temperature (90 °C), and K m values of 0.1, 10 and 50 mM for PNP-β-glucoside, laminaribiose and cellobiose, respectively. The gene bgIB was located immediately upstream of the laminarinase gene IamA. Both genes were transcribed from the same DNA strand and were not separated by a palindromic transcription terminator. The two purified enzymes 1,3-β-glucosidase BgIB (laminaribiase) and 1,3-β-glucanase LamA (laminarinase) were together capable of completely degrading laminarin to glucose.

The authors previously reported the isolation and partial characterization of a periplasmically located dihydrolipoamide dehydrogenase (LPD) from the cyanobacterium Synechocystis sp. strain PCC 6803. In the present work the gene (IpdA; database accession number Z48564) encoding the apoprotein of this LPD in Synechocystis PCC 6803 has been identified, sequenced and analysed. The IpdA gene codes for a protein starting with methionine, which is post-translationally removed. The mature protein contains an N-terminal serine and consists of 473 amino acids with a deduced molecular mass of 51421 Da (including one FAD). The LPD is an acidic protein with a calculated isoelectric point of 5.17. Comparison of the amino acid sequence of the Synechocystis LPD with protein sequences in the databases revealed that the enzyme shares identities of 31-35% with all 18 LPDs so far sequenced and published. As a first step in determining the role of this cyanobacterial LPD, attempts were made to generate an LPD-free Synechocystis mutant by insertionally inactivating the IpdA gene with a kanamycin-resistance cassette. However, the selected transformants appeared to be heteroallelic, containing both the intact IpdA gene and the IpdA gene inactivated by the drug-resistance cassette. The heteroallelic mutant studied, which had about 50% of the wild-type LPD activity, caused acidification of the growth medium. Growth over a prolonged time was only possible after an increased buffering of the medium. Since it is reported in the literature that inactivation of the pyruvate dehydrogenase complex (PDC) leads to acidosis, a function of the LPD in a cytoplasmic-membrane-associated PDC is conceivable.

In previous experiments, the Streptomyces aureofaciens gap gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified. To investigate expression of the gene, S1 nuclease mapping and Northern blot hybridization were performed using RNA prepared from S. aureofaciens cultivated under various conditions. These studies suggested monocistronic organization and developmental regulation of the gene. A single promoter, gap-P, was identified upstream of the gap coding region. In cultures grown on solid medium in the absence of glucose, its transcription was induced at the time of aerial mycelium formation. In addition, gap transcription was also induced in substrate mycelium by glucose. A promoter-bearing DNA fragment was inserted into two promoter-probe vectors, to give expression patterns consistent with the results of direct RNA analysis.

An initiator tRNA gene, metA, and a closely linked fragment of a second initiator-tRNA-like sequence, metB, from Mycobacterium tuberculosis H37Ra have been cloned and characterized. The promoter region of metA shows the presence of conserved sequence elements, TAGCCT and TTGGCG, with resemblance to -10 and -35 promoter regions. The deduced sequence of the mature tRNA contains the three unique features of the eubacterial initiator tRNAs represented by (i) a C:U mismatch at position 1:72, (ii) three consecutive base pairs, 29-31G:C39-41 in the anticodon stem, and (iii) a purine:pyrimidine (A:U) base pair at position 11:24 in the dihydrouridine stem. A putative hairpin structure consisting of an 11 bp stem and a three-base loop found in the 3′ flanking region is followed by a stretch of T residues and may serve as a transcription terminator. Analysis of the expression of metA and of its promoter using chloramphenicol acetyltransferase fusion constructs in Mycobacterium smegmatis shows that metA is a functional gene driven by a strong promoter. Furthermore, the overexpressed transcripts are fully processed and formylated in vivo. The metB clone shows the presence of sequences corresponding to those downstream of position 30 of the tRNA. However, the CCA sequence at the 3′ end has been mutated to CCG. Interestingly, the 3′ flanking sequences of both the genes are rich in GCT repeats. The metB locus also harbours a repeat element, IS6110. A method to prepare total RNA from mycobacteria (under acidic conditions) to analyse in vivo status of tRNAs is described.