- Volume 149, Issue 2, 2003

This work characterized the putative quinone oxidoreductase gene (qorA) from Staphylococcus aureus. The deduced amino acid sequence indicated that the 333 aa protein contains an NAD(P)H-binding motif. A Northern blot analysis revealed that 2·6 kb and 1·4 kb signals were detected by using a qorA probe. Both the signals were enhanced under the presence of a redox-cycling agent, 9,10-phenanthrenequinone (PQ). It was also revealed that the expression of three genes, SA1988, SA1989 (qorA) and SA1990, was enhanced at the transcriptional level by PQ exposure. The results suggested that the 2·6 kb signal detected by the qorA probe was in two co-transcripts, i.e. SA1990–qorA and qorA–SA1988 were transcribed. Besides, primer extension analyses confirmed the enhancement of qorA and SA1990 transcripts. The GST (glutathione S-transferase)-tagged QorA protein was expressed in Escherichia coli and purified using a glutathione affinity column. In purification steps, a 36 kDa band co-purified with the GST–QorA, and it was detected even in the thrombin-cleaved fraction. N-terminal amino acid sequences for the 36 kDa protein revealed that it was an intact QorA. They showed that QorA formed a multimer under physiological conditions. The purified recombinant GST–QorA catalysed NADPH consumption in the presence of PQ as a substrate, but not NADH. To characterize the catalytic activity of QorA, superoxide anion that was generated through one-electron reduction of PQ and hydroquinone that was produced by two-electron reduction of PQ were measured. During reduction of PQ by GST–QorA, superoxide anion was generated, whereas a small amount of 9,10-dihydroxyphenanthrene (hydroquinone of PQ) was produced. These results suggest that the activity of QorA is similar to ζ-Crystallin, catalysing an NADPH-dependent one-electron reduction of quinone.

The Streptococcus gordonii glucosyltransferase structural gene, gtfG, is located immediately downstream from its positive transcriptional regulatory determinant, rgg. Recent genetic studies have indicated that the 3′ end of rgg is involved either directly as a binding site or indirectly, e.g. by playing a role in secondary structure, in the interaction of Rgg with the gtfG promoter. A previously identified spontaneous mutant with a point mutation near the 3′ end of rgg had only ∼25 % of the parental level of glucosyltransferase activity. To determine if this decreased activity was due to a change in the DNA binding site of trans-acting Rgg, or due to a change in the Rgg protein itself, complementation analyses and DNA-binding studies were performed. In Rgg-deficient strains, the chromosomal rgg point mutation did not influence the ability of plasmid-borne rgg to increase glucosyltransferase expression. However, plasmids carrying parental rgg were able to increase glucosyltransferase activity and expression of a gtfG promoter fusion to a greater extent than plasmids carrying the mutant allele, indicating that the mutant Rgg protein had decreased activity. The ability of NH2-terminal (hexahistidine) tagged proteins to bind to a 107 bp dsDNA fragment corresponding to the region immediately upstream of gtfG was demonstrated by surface plasmon resonance. Despite their differences in activity, both mutant and parental recombinant Rgg proteins bound to this dsDNA, albeit with different strengths. These studies provide insights into functional domains of S. gordonii Rgg which influence glucosyltransferase expression, and may have implications for Rgg-like regulatory proteins in related bacteria.

The main causes of microbial death after heat exposure are not well understood. Here, it is shown that the heat-shock protein ClpP plays a major role in heat-induced growth arrest in Streptococcus agalactiae. A mutant lacking the ClpP protease was more sensitive to the inhibitory effects of heat, salt and oxidative stress than the isogenic wild-type strain. During growth arrest, this mutant displayed important modifications of its total protein content, including a decreased level of essential metabolic enzymes such as the alcohol dehydrogenase. Analysis of protein carbonylation demonstrated that the ClpP protease plays a role in preventing accelerated protein oxidation. Higher levels of oxidized DnaK, a key modulator of the heat-shock regulon, were observed in the ClpP mutant and these were increased following heat shock. Accumulation of oxidized/inactivated DnaK might explain why the ClpP mutant was unable to properly synthesize DNA and proteins, and why it exhibited an aberrant cell morphology. Even though ClpP plays a minor role in the virulence of S. agalactiae in a murine infection model, the data presented here point to the importance of ClpP in oxidative stress defence in preventing heat-induced cell alterations.

Methylobacillus sp. strain 12S produces an exopolysaccharide (EPS), methanolan, composed of glucose, mannose and galactose. Twenty-four ORFs flanking a Tn5 insertion site in an EPS-deficient mutant were identified, and 21 genes (epsCBAKLDEFGHIJMNOPQRSTU) were predicted to participate in methanolan synthesis on the basis of the features of the primary sequence. Gene disruption analyses revealed that epsABCEFGIJNOP and epsR are required for methanolan synthesis, whereas epsKD and epsH are not essential. EpsFG and EpsE showed homology with Wzc (chain length regulator) and Wza (export protein) of group 1 capsule-producing Escherichia coli, suggesting that methanolan was synthesized via a Wzy-like biosynthesis system. This possibility was supported by the fact that the putative hydropathy profiles of EpsH and EpsM were similar to those of Wzx and Wzy, which are also involved in the flipping of the repeating unit in the cytoplasmic membrane and the polymerization of the capsule in the Wzy-dependent system. EpsBJNOP and EpsR are probably glycosyltransferases involved in the synthesis of the repeating unit onto the lipid carrier. In particular, EpsB appeared to catalyse the initial transfer of the glucose moiety. On the basis of their predicted location in the cells, it is proposed that EpsI and EpsL are involved in methanolan export to the cell surface. E. coli strains expressing EpsQ, EpsS and EpsT showed enhanced activities of GDP-mannose pyrophosphorylase, UDP-galactose 4-epimerase and UDP-glucose pyrophosphorylase, respectively, revealing that they were responsible for the production of the activated compositional sugars of methanolan. EpsU contains a conserved a lytic transglycosylase motif, indicating that it could participate in the degradation of polysaccharides. EpsA and EpsK, which have conserved DNA-binding and cAMP-binding motifs, respectively, were deduced to be transcriptional regulators. In particular, EpsA seems to positively regulate the transcription of methanolan synthesis genes, since the constitutive expression of epsA in strain 12S increased the EPS production. Interestingly, EpsD showed homology with peptidyl prolyl cis–trans isomerases that catalyse the folding of proteins following translocation across the cytoplasmic membrane.

Azospirillum lipoferum RG20, a nitrogen-fixing bacterium found in all kind of soils, was found to be naturally resistant to penicillins and cephalosporins. 6-β-Bromopenicillanic acid, an irreversible inhibitor of serine-β-lactamases, completely abolished this resistance. A β-lactamase was purified 518-fold from a cell-free extract of A. lipoferum RG20. A single band on SDS-PAGE (apparent molecular mass 31 000 Da) and on isoelectric focussing (pI9·35) was observed with the purified protein. The enzyme hydrolysed benzylpenicillin, ampicillin, cephalothin and cephaloridine with comparable k cat values and catalytic efficiencies. However, carbenicillin and cefotaxime were hydrolysed with significantly lower kinetic parameters and oxacillin was hydrolysed at a rate 100 times slower. The purified β-lactamase was inhibited by clavulanic acid and sulbactam but not by EDTA or aztreonam. Its substrate and inhibitor profiles are consistent with those of the broad-spectrum β-lactamases inhibited by clavulanic acid (group 2b of the Bush–Jacoby–Medeiros scheme). The effect of pH on k cat and K m values for benzylpenicillin hydrolysis was studied. The dependence of k cat on pH suggests that the enzyme–substrate (ES) complex must be in at least three protonation states: two with k cat values equal to 2800 and 1450 s−1 and a third inactive one [pK 1(ES) 4·7 and pK 2(ES) 7·9]. Similarly, the dependence of k cat/K m on pH can be explained by postulating that the enzyme free form can be at least in three different protonation states: two of them with k cat/K m values equal to 2·7×106 and 3·7×108 M−1 s−1 and a third one unable to productively bind substrate. Interestingly, the dependence of k cat/K m on pH is consistent with positive cooperativity for proton binding to the enzyme free form [pK 1(E) 8·5 and pK 2(E) 7·2].

The albA gene from Klebsiella oxytoca encodes a protein that binds albicidin phytotoxins and antibiotics with high affinity. Previously, it has been shown that shifting pH from 6 to 4 reduces binding activity of AlbA by about 30 %, indicating that histidine residues might be involved in substrate binding. In this study, molecular analysis of the albA coding region revealed sequence discrepancies with the albA sequence reported previously, which were probably due to sequencing errors. The albA gene was subsequently cloned from K. oxytoca ATCC 13182T to establish the revised sequence. Biochemical and molecular approaches were used to determine the functional role of four histidine residues (His78, His125, His141 and His189) in the corrected sequence for AlbA. Treatment of AlbA with diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, reduced binding activity by about 95 %. DEPC treatment increased absorbance at 240–244 nm by an amount indicating conversion to N-carbethoxyhistidine of a single histidine residue per AlbA molecule. Pretreatment with albicidin protected AlbA against modification by DEPC, with a 1 : 1 molar ratio of albicidin to the protected histidine residues. Based on protein secondary structure and amino acid surface probability indices, it is predicted that His125 might be the residue required for albicidin binding. Mutation of His125 to either alanine or leucine resulted in about 32 % loss of binding activity, and deletion of His125 totally abolished binding activity. Mutation of His125 to arginine and tyrosine had no effect. These results indicate that His125 plays a key role either in an electrostatic interaction between AlbA and albicidin or in the conformational dynamics of the albicidin-binding site.

N-Acyl-l-homoserine-lactone-producing Serratia species are frequently encountered in spoiling foods of vegetable and protein origin. The role of quorum sensing in the food spoiling properties of these bacteria is currently being investigated. A set of luxR luxI homologous genes encoding a putative quorum sensor was identified in the N-(3-oxo-hexanoyl)-l-homoserine lactone (3-oxo-C6-HSL)-producing Serratia proteamaculans strain B5a. The 3-oxo-C6-HSL synthase SprI showed 79 % similarity with EsaI from Pantoea stewartii and the putative regulatory protein SprR was 86 % similar to the SpnR of Serratia marcescens. Proteome analysis suggested that the presence of at least 39 intracellular proteins was affected by the 3-oxo-C6-HSL-based quorum sensing system. The lipB-encoded secretion system was identified as one target gene of the quorum sensing system. LipB was required for the production of extracellular lipolytic and proteolytic activities, thus rendering the production of food-deterioration-relevant exoenzymes indirectly under the control of quorum sensing. Strain B5a caused quorum-sensing-controlled spoilage of milk. Furthermore, chitinolytic activity was controlled by quorum sensing. This control appeared to be direct and not mediated via LipB. The data presented here demonstrate that quorum-sensing-controlled exoenzymic activities affect food quality.

Capsule phase variants were isolated from serotype 8 and serotype 37 pneumococcal sorbarods. Sequence duplications within the essential capsule genes – cap8E (type 8) and tts (type 37) – were found to introduce frameshifts and generate acapsular phenotypes. Capsular revertants possessed wild-type cap8E and tts genes, indicating the precise excision of these duplications. Reversion frequencies (OFF–ON) fit a linear relationship between log(frequency of reversion) and log(length of duplication), previously found for serotype three pneumococci [ Waite, R. D., Struthers, J. K. & Dowson, C. G. (2001) . Mol Microbiol 42, 1223–1232]. This study provides evidence that capsule phase variation can occur in pneumococcal serotypes with either simple (one to three genes) or complex capsule-encoding loci (12 genes). Given the key role of CapE (the first monosaccharide transferase) in other clinically important pneumococci, such as serotypes 14 and 19F with complex capsular loci, the observed duplication within cap8E suggests that capsule phase variation could be controlled by tandem sequence duplication in capE homologues in other pneumococcal serotypes that construct their capsules through polymerization of lipid-linked intermediates.

Plasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the λ integrase family of site-specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1·6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix–turn–helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcriptional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome.

Clostridium thermocellum produces a great number of extracellular cellulases which are free or cellulosome-bound. The nucleotide sequence of a gene cluster containing the genes celI, celN and cseP was determined from C. thermocellum strain F7. Gene products Cel9I and Cel9N are structurally related enzymes having a glycosyl hydrolase family 9 and a carbohydrate-binding module (CBM3c), but show characteristic differences: Cel9I is a non-cellulosomal protein with an additional CBM (CBM3b), whereas Cel9N contains a cellulosomal dockerin module and no additional CBM. Although Cel9I is a processive endoglucanase, Cel9N is non-processive. Both enzymes hydrolyse phosphoric acid swollen cellulose, but the products of hydrolysis are different. The CseP protein encoded in the gene cluster is the first component attached to the cellulosomal scaffoldin for which no catalytic activity could be detected. It was shown to be present in the cellulosome. Its sequence is homologous to the spore-coat assembly protein CotH of Bacillus subtilis, suggesting a structural role of CseP in the cellulosome.

The homologous CsgD and AgfD proteins are members of the FixJ/UhpA/LuxR family and are proposed to regulate curli (thin aggregative fibres) and cellulose production by Escherichia coli and Salmonella enterica serovar Typhimurium, respectively. A plasmid containing part of the csgD gene was isolated during a screen for multicopy suppressors of glycine auxotrophy caused by deleting the folA gene in E. coli. The sequence of the plasmid suggests it encodes a chimaeric protein. Plasmids containing the intact csgD or agfD gene also caused suppression. Cells transformed with the recombinant plasmids contained higher serine hydroxymethyltransferase (SHMT) activity than controls. The increase could also be monitored by assaying β-galactosidase activity from a reporter strain with part of the SHMT gene, glyA, fused to lacZ. The increase in SHMT activity was sufficient to correct the glycine auxotrophy of strains lacking folA. The recombinant plasmids also enabled K-12 strains that are not curli-proficient to make curli. Curlin, the major component of curli, contains more glycine than normal E. coli proteins. It is proposed that CsgD upregulates glyA to facilitate synthesis of curli. It is suggested that recombinant plasmids produce enough CsgD or chimaeric protein to titrate out a ligand that switches CsgD into its inactive form. As a result, sufficient active CsgD is present to activate genes in its regulon. It is concluded that CsgD increases expression of the glyA gene either directly or indirectly.

Mimosine is a toxin present in the tree-legume leucaena (Leucaena leucocephala), including its root nodules and the root exudates. The leucaena-nodulating Rhizobium sp. strain TAL1145 degrades mimosine (Mid+) and utilizes it as a source of carbon and nitrogen. Twelve TAL1145 mutants defective in mimosine degradation (Mid−) were made through Tn3Hogus, TnphoA or kanamycin-resistance-cassette insertions. A 5·0 kb PstI fragment of TAL1145, subcloned from a cosmid clone containing mid genes for mimosine degradation, complemented most of the Mid− mutants. Sequencing this fragment and the adjacent 0·9 kb PstI fragment identified five genes, midA, midB, midC, midD and midR, of which the first three genes encode ABC transporter proteins involved in mimosine uptake, while midD encodes an aminotransferase required for degrading mimosine into 3-hydroxy-4-pyridone, and midR is a regulatory gene encoding a LysR-type transcriptional activator. The location of MidA in the periplasm was shown by making two midA : : phoA fusions, which made active alkaline phosphatase in the periplasm. The various mid : : gus and midA : : phoA fusions were inducible by mimosine, and a midD : : gus fusion mutant showed β-glucuronidase activity in the leucaena nodules, indicating that midD is expressed in the nodules. Similarly, a midA : : phoA fusion expressed alkaline phosphatase activity in the leucaena nodules, indicating that mimosine induces midA transcription in the bacteroids. mid genes are specific for the Mid+ strains of leucaena Rhizobium and are absent in strains of other Rhizobium, Sinorhizobium and Bradyrhizobium spp.