- Volume 144, Issue 3, 1998

Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and ‘Tissierella creatinophila’ based upon their properties: the binding to 2′,5′-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.

FNR (regulator for fumarate and nitrate reduction) and CRP (cAMP receptor protein) are global regulators which regulate the transcription of overlapping modulons of target genes in response to anaerobiosis and carbon source in Escherichia coli. An ORF, designated flp because it encodes an FNR-like protein of the FNR-CRP family, has been found in Lactobacillus casei. The product of the flp coding region (FLP) was overproduced in E. coli, purified and crystallized. FLP is a homodimeric protein in which each subunit can form an intramolecular disulphide bond. The isolated protein also contains non-stoichiometric amounts of Cu and Zn. Although the DNA recognition helix of FLP resembles that of FNR, the flp gene failed to complement the anaerobic respiratory deficiency of an fnr mutant when expressed in E. coli and it neither activated nor interfered with transcription from FNR- or CRP-dependent promoters in E. coli. Site-specific DNA binding by oxidized FLP (the form containing intrasubunit disulphide bonds) was abolished by reduction. The interconversion between disulphide and dithiol forms thus provides the basis for a novel redox-mediated transcriptional switch. Two non-identical FLP-binding sites, distinct from FNR- and CRP-binding sites, were identified in the meIR region of E. coli by gel-retardation analysis. A further eight FLP-binding sites were selected from a random library. A synthetic oligonucleotide conforming to a putative FLP site consensus, CA/cTGA-N4-TCAG/TG (the most significant bases are underlined), was retarded by FLP. Functional tests showed that FLP represses the aerobic transcription of a semi-synthetic promoter in E. coli. A C5S variant of FLP lacking the ability to form intramolecular disulphide bonds was unable to bind to FLP sites and failed to repress transcription in vivo.

Lactobacillus helveticus CNRZ 892 contains a surface layer (S-layer) composed of protein monomers of 43 kDa organized in regular arrays. The gene encoding this protein (sIpH) has been cloned in Escherichia coli and sequenced. sIpH consists of 440 codons and is preceded by a ribosome-binding site (RBS) and followed by a putative ρ-independent terminator. Indeed, Northern analysis revealed that sIpH is a monocistronic gene. The gene is preceded by a possible promoter of which the -35 and -10 hexanucleotides are separated by 17 nt. By primer extension analysis the transcription start site was mapped at 7 nt downstream of the -10 sequence while the deduced amino acid sequence of SIpH shows a leader peptide of 30 aa. The sIpH gene has been amplified by PCR and the fragment, carrying the complete gene from the RBS to the stop codon, has been cloned in a lactococcal gene expression vector downstream of promoter P32. Lactococcus lactis MG1363 carrying the resulting plasmid produced and secreted an S-layer monomer with the same molecular mass as the authentic L. helveticus CNRZ 892 SIpH, as judged by SDS-PAGE. Immunoelectron microscopy revealed that SIpH was bound to the lactococcal cell walls in small clumps and accumulated in the growth medium as small sheets.

In Streptomyces coelicolor A3(2), bldA mutants that lack the tRNA for the rare leucine codon UUA fail to make the red undecylprodigiosin antibiotic complex. To find out why, red-pigmented while bald (Pwb) derivatives of a bldA mutant were isolated. Using a cloning strategy that allowed for (and demonstrated) dominance of the mutations, they were localized to the red gene cluster. By using insert-mediated integration of a ?C31 phage-based vector, one of the Pwb mutations was more precisely located between red structural genes to a segment of approximately 1 kb about 4 kb from the known pathway-specific regulatory gene redD. The segment contained most of an ORF (redZ) encoding a protein (RedZ) with end-to-end similarity to response regulators of diverse function from a variety of bacteria. Remarkably, in RedZ hydrophobic residues replace nearly all of the charged residues that usually make up the phosphorylation pocket present in typical response regulators, including the aspartic acid residue that is normally phosphorylated by a cognate sensory protein kinase. A single TTA codon in redZ provided a potential explanation for the bldA-dependence of undecylprodigiosin synthesis. This codon was unchanged in three Pwb mutants, but further analysis of one of the mutants revealed a potential up-promoter mutation. It seems possible that a combination of low-level natural translation of the UUA codon by a charged non-cognate tRNA, coupled with increased transcription of redZ in the Pwb mutant, allows the accumulation of a threshold level of the RedD protein.

The functional stability of the chloramphenicol acetyltransferase (cat) mRNA, as well as the functional stability of the total mRNA pool, change during the course of Escherichia coli culture growth. mRNA half-lives are long during lag phase, decrease during the exponential phase and increase again during the stationary phase of the bacterial growth cycle. The half-lives of cat mRNA and total mRNA also increase three- to fourfold during amino acid starvation when compared to exponential culture growth. Even though the stability of the cat message changes about fourfold during culture growth, the amount of cat mRNA per cell mass does not vary significantly between the culture growth phases, indicating that there are compensating changes in cat gene transcription. Translation of cat mRNA also changes during culture growth. In exponential phase, the rate of cat translation is about 14-fold higher than when the culture is in stationary phase. This is in contrast to the fourfold increase in stability of cat mRNA in the stationary-phase culture compared to the exponentially growing culture and indicates that active translation is not correlated with increased mRNA stability. When a stationary-phase culture was diluted into fresh medium, there was a five- to sevenfold increase in CAT synthesis and a threefold increase in total protein synthesis in the presence or absence of rifampicin. These results suggest that while mRNA becomes generally more stable and less translated in the stationary-phase culture, the mRNA is available for immediate translation when nutrients are provided to the culture even when transcription is inhibited.

Certain amino acids are substrates for two decarboxylase enzymes in Escherichia coli, one inducible by anaerobic growth at low pH and the other constitutive. In the case of lysine, an inducible decarboxylase (CadA) has been extensively characterized, but evidence for the existence of a second lysine decarboxylase is fragmentary and uncertain. This paper confirms that a second lysine decarboxylase is encoded by a locus (Idc) previously suggested to be a lysine decarboxylase gene on the basis of sequence comparisons. Overexpression of the cloned gene provided sufficient quantities of enzyme in cell-free extracts for preliminary examination of the properties of the Idc gene product, Ldc. The enzyme is active over a broad range of pH with an optimum at 7.6, much higher than that of CadA, about 5.5. The temperature optimum for both enzymes is similar, at about 52 °C, but Ldc is more readily inactivated by heat than CadA. Expression of Idc from its own promoter was very weak for cells growing in a variety of media, although a low level of lysine decarboxylase was present in cells that carried the Idc region on an oligo-copy plasmid when these were grown in minimal-glucose medium. Northern analysis of RNA extracted from such cells revealed a transcript whose length corresponded to that of the ldc gene, suggesting that ldc is normally transcribed from a promoter immediately upstream. However, most of the ldc mRNA was shorter, indicating degradation or premature termination. The ldc upstream sequence promoted transcription of a lacZ gene to which it was fused. Introduction of the upstream sequence as an insert in a multicopy vector increased transcription of the resident lacZ fusion. The low level of expression in single copy, the emergence of expression when the gene is present at moderate copy number, and the derepression by the upstream sequence in trans imply that this second lysine decarboxylase gene may not be constitutive but subject to specific repression by a factor which remains to be identified.

A protein similar to the previously characterized variable surface-exposed membrane protein P120 was identified (P120′), establishing that Mycoplasma hominis PG21 possesses a novel gene family. The gene, p120', was sequenced and found to have some distinctive properties including a putative start codon of GTG, rather than the common ATG codon, and a coding region with a high G+C content, characteristic of essential housekeeping genes in mycoplasmas. No sequence homology was found to known proteins. The genomic locations of the p120 and p120' genes were determined on the restriction map of five M. hominis strains by PFGE. The genes were localized in two separate regions separated by more than 6 kb. Genes as well as proteins corresponding to P120′ were identified in 24/24 M. hominis isolates tested and no size variation was detected. P120′ had a molecular mass of 98 kDa, 20 kDa smaller than P120 as estimated by SDS-PAGE. The protein was surface-exposed and associated with the mycoplasma membrane, but had predominantly hydrophilic characteristics upon Triton X-114 extraction. The N-terminal part of P120′ had a hydrophobic leader sequence without the characteristics of a prolipoprotein. This might explain the membrane association of the protein. Unlike P120, which is frequently recognized by sera of patients seropositive for M. hominis, P120′ was only rarely recognized. The conserved nature of the P120 gene family indicates that it has an essential, although currently unknown, function.

The distribution of four classes of related plasmid replication genes (repC) within three field populations of Rhizobium leguminosarum in France, Germany and the UK was investigated using RFLP, PCR-RFLP and plasmid profile analysis. The results suggest that the four repC classes are compatible: when two or more different repC sequences are present in a strain they are usually associated with different plasmids. Furthermore, classical incompatibility studies in which a Tn5-labelled plasmid with a group IV repC sequence was transferred into field isolates by conjugation demonstrated that group IV sequences are incompatible with each other, but compatible with the other repC groups. This supports the idea that the different repC groups represent different incompatibility groups. The same field isolates were also screened for chromosomal (plac12) and symbiotic gene (nodD-F region) variation. Comparison of these and the plasmid data suggest that plasmid transfer does occur within field populations of R. leguminosarum but that certain plasmid-chromosome combinations are favoured.

The genetic diversity of clinical isolates of Streptococcus pyogenes serotype M5 has been characterized. Strain genotypes were defined by macrorestriction profile, 16S ribotype, emm gene subtype, insertion element IS 1239 profile, and exotoxin gene determinant. By these criteria, clinical isolates of M5 constituted a multiplicity of strain clusters rather than a homogeneous population as found for certain serotypes. Distance matrices and an unrooted tree were constructed from macrorestriction data with three rarely cutting endonucleases, determined by PFGE. A single IS1239 profile was common to 85% of isolates but there was great diversity of both ribotype and macrorestriction profile, and 18 different emm gene subtypes were detected by PCR-RFLP. DNA sequence analysis of the antigen-coding 5' (hypervariable) region of emm gene amplicons (about 240 bp) showed that 14/18 exhibited up to 6% divergence. Four amplicons had highly divergent sequences - corresponding to those previously determined for emm 6, emm 11, emm 18 and emm 77. Further serological and hybridization studies were used to analyse the discrepancy between the Lancefield serotype of these strains (W5) and their emm genotype. Overall, this study shows a high degree of genetic diversity in serotype M5, with implications for the Lancefield scheme itself, for the epidemiology of group A streptococci, and for recombinant DNA strategies for M protein-based vaccine development.

Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress. In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress. The survival under severe osmotic stress (0.866 a W, NaCI or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid-exponential or the stationary growth phase on glucose, galactose or ethanol. Stationary-phase cells survived better than exponential-phase cells. The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells. There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress. The mutant strains unable to produce trehalose (tps1Δ tps2Δ and tps1Δ hxk2 Δ) were more sensitive to severe osmotic stress (0.866 a W) than the isogenic wild-type strain, confirming a role for trehalose in survival. Hyperaccumulation of trehalose found in the nth1Δ and the nth1Δ gpd1Δ mutant strains, however, did not improve survival rates compared to the wild-type strain. When wild-type, nth1Δ and nth1Δ gpd1Δ cells were exposed to moderate osmotic stress (0.98 and 0.97 a W, NaCI), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1Δ cells than in wild-type cells during the initial response to osmotic stress. These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions. No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S. cerevisiae under osmotic stress conditions.