- Volume 148, Issue 11, 2002

Previous work indicated that Streptococcus bovis HC5 had significant antibacterial activity, and even nisin-resistant S. bovis JB1 cells could be strongly inhibited. S. bovis HC5 inhibited a variety of Gram-positive bacteria and the spectrum of activity was similar to monensin, a commonly used feed additive. The crude extracts (ammonium sulfate precipitation) were inactivated by Pronase E and trypsin, but the activity was resistant to heat, proteinase K and α-chymotrypsin. Most of the antibacterial activity was cell associated, but it could be liberated by acidic NaCl (100 mM, pH 2·0) without significant cell lysis. When glycolysing S. bovis JB1 cells were treated with either crude or acidic NaCl extracts, intracellular potassium declined and this result indicated the antibacterial activity was mediated by a pore-forming peptide. The peptide could be purified by HPLC and matrix-assisted laser desorption ionization time-of-flight analysis indicated that it had a molecular mass of approximately 2440 Da. The terminal amino acid sequence was VGXRYASXPGXSWKYVXF. The unnamed amino acid residues (designated by X) had approximately the same position as dehydroalanines found in some lantibiotics, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. The only other bacteriocin that had significant similarity was the lantibiotic precursor of Streptococcus pyogenes SF370, but the identity was only 55%. Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and the authors now designate it as bovicin HC5.

Laboratory-scale sequencing batch reactors (SBRs) as models for wastewater treatment processes were used to identify glycogen-accumulating organisms (GAOs), which are thought to be responsible for the deterioration of enhanced biological phosphorus removal (EBPR). The SBRs (called Q and T), operated under alternating anaerobic–aerobic conditions typical for EBPR, generated mixed microbial communities (sludges) demonstrating the GAO phenotype. Intracellular glycogen and poly-β-hydroxyalkanoate (PHA) transformations typical of efficient EBPR occurred but polyphosphate was not bioaccumulated and the sludges contained 1·8% P (sludge Q) and 1·5% P (sludge T). 16S rDNA clone libraries were prepared from DNA extracted from the Q and T sludges. Clone inserts were grouped into operational taxonomic units (OTUs) by restriction fragment length polymorphism banding profiles. OTU representatives were sequenced and phylogenetically analysed. The Q sludge library comprised four OTUs and all six determined sequences were 99·7% identical, forming a cluster in the γ-Proteobacteria radiation. The T sludge library comprised eight OTUs and the majority of clones were Acidobacteria subphylum 4 (49% of the library) and candidate phylum OP10 (39% of the library). One OTU (two clones, of which one was sequenced) was in the γ-Proteobacteria radiation with 95% sequence identity to the Q sludge clones. Oligonucleotide probes (called GAOQ431 and GAOQ989) were designed from the γ-Proteobacteria clone sequences for use in fluorescence in situ hybridization (FISH); 92% of the Q sludge bacteria and 28% of the T sludge bacteria bound these probes in FISH. FISH and post-FISH chemical staining for PHA were used to determine that bacteria from a novel γ-Proteobacteria cluster were phenotypically GAOs in one laboratory-scale SBR and two full-scale wastewater treatment plants. It is suggested that the GAOs from the novel cluster in the γ-Proteobacteria radiation be named ‘Candidatus Competibacter phosphatis’.

Antibiotic production in Streptomyces lividans can be activated by introducing certain mutations (rif) into the rpoB gene that confer resistance to rifampicin. Working with the most typical (rif-17) mutant strain, KO-417, the rif-17 mutation was characterized. The rif-17 mutation was shown to be responsible for activating antibiotic production and for reducing the growth rate of strain KO-417, as demonstrated by gene-replacement experiments. Gene-expression analysis revealed that introduction of rif into S. lividans elevates expression of the pathway-specific regulatory gene actII-ORF4 to nearly the same level seen in Streptomyces coelicolor. The rif effect on antibiotic production was still evident in the genetic background of relC, indicating that the rif mutation can provoke its effect without depending on ppGpp. Accompanying the restoration of antibiotic production, rif mutants also exhibited a lower rate of RNA synthesis compared to the parental strain when grown in a nutritionally rich medium, suggesting that the mutant RNA polymerases may behave like ’stringent’ RNA polymerases. These results indicate that the rif mutation can alter the gene-expression pattern independent of ppGpp. The impaired growth of strain KO-417 (rif-17) was largely restored by introducing the second rif mutation (rif-18) just adjacent to the rif-17 position. Proteome analysis using two-dimensional PAGE revealed that the rif mutant strain KO-418 (rif-17 rif-18) displayed a temporal burst of expression especially of two enzymes, glutamine synthetase (type II) and oxidoreductase, during the late growth phase.

In this study a set of Streptomyces galilaeus ATCC 31615 mutants was characterized, which are incapable of synthesizing some or all of the deoxyhexose sugars of aclacinomycin A. Complementation experiments with the the mutant strains H026, H038, H039, H054, H063, H065 and H075 were carried out with glycosylation genes previously derived from the wild-type S. galilaeus. Mutations in strains H038, H063 and H075 were complemented with single PCR-amplified genes. Furthermore, amplification and sequencing of the corresponding genes from the mutant strains revealed single point mutations in the sequences. First, in H038 a transition mutation in aknQ, encoding a putative dTDP-hexose 3-ketoreductase, causes an amino acid substitution from glycine to aspartate, suppressing the biosynthesis of both 2-deoxyfucose and rhodinose and thus leading to the accumulation of aclacinomycin T with rhodosamine as its only sugar. Second, in H063, which accumulates aklavinone without a sugar moiety, amino acid substitution occurs, with threonine being substituted by isoleucine in dTDP-glucose synthase, the first enzyme participating in deoxyhexose biosynthesis, encoded by aknY. Third, a nonsense mutation in aknP leads to truncated dTDP-hexose 3-dehydratase in H075, which is incapable of synthesizing rhodinose. In addition, mutants H054 and H065, which accumulate aclacinomycins without aminosugars, were complemented by a gene for an aminotransferase, aknZ. Characterization of the nature of the mutations adds to the usefulness and value of the mutants in the analysis of gene function and in the creation of novel compounds by combinatorial biosynthesis. Furthermore, these results strengthen the assignments of akn gene products and enlighten the biosynthetic pathway for deoxyhexoses.

The glucosyltransferases of Streptococcus mutans are recognized as important virulence factors for this cariogenic bacterium. To study the expression of the gtfB gene of S. mutans in biofilms, a gtfB promoter (PgtfB)–green fluorescent protein (GFP) reporter system was developed. A Streptococcus–Escherichia coli shuttle vector harbouring a PgtfB::gfp cassette was introduced into S. mutans GS-5, and the expression of GFP by the transformed S. mutans cells was confirmed by fluorescence microscopy. Furthermore, confocal laser scanning microscopy was carried out on biofilms attached to polystyrene plates; enhanced gtfB expression was observed in various microcolonies across these biofilms. To further test the hypothesis that gtfB expression is upregulated in biofilms, flow cytometry analysis was done on planktonic and biofilm cells; this analysis showed an approximately five-fold increase in gtfB expression in the biofilm cells relative to the planktonic cells. Real-time (TaqMan) PCR analysis confirmed that gtfB expression in the biofilm cells was enhanced relative to the planktonic cells. Previously, it has been suggested that the S. mutans gtfC gene might be co-transcribed with gtfB. Therefore, RT-PCR analysis was performed on gtfB-expressing S. mutans; this analysis demonstrated that gtfC was co-transcribed with gtfB. These results indicated that GFP expression can be utilized to examine gene regulation in S. mutans biofilm formation.

Native and recombinant FomA proteins were extracted by detergent from the cell envelopes of Fusobacterium nucleatum and Escherichia coli, and purified to near homogeneity by chromatography. Circular dichroism analysis revealed that the FomA protein consists predominantly of β-sheets, in line with the previously proposed 16-stranded β-barrel topology model. Results obtained by trypsin treatment of intact cells and cell envelopes of F. nucleatum, and from limited proteolysis of purified FomA protein, indicated that the N-terminal part of the FomA protein is not an integral part of the β-barrel, but forms a periplasmic domain. Based on these results a new topology model is proposed for the FomA protein, where the C-terminal part forms a 14-stranded β-barrel separate from the periplasmic N-terminal domain.

Bacillus subtilis 168 was assayed for its growth on tricarboxylic acid (TCA) cycle intermediates and related compounds as the sole carbon sources. Growth of the organism was supported by citrate, D-isocitrate, succinate, fumarate and L-malate, whereas no growth was observed in the presence of cis-aconitate,2-oxoglutarate, D-malate, oxaloacetate and tricarballylate. Growth of the organism on the tricarboxylates citrate and D-isocitrate required the presence of functional CitM, an Mg2+–citrate transporter, whereas its growth on succinate, fumarate and L-malate appeared to be CitM-independent. Interestingly, the naturally occurring enantiomer D-isocitrate was favoured over L-isocitrate by the organism. Like citrate, D-isocitrate was shown to be an inducer of citM expression in B. subtilis. The addition of 1 mM Mg2+ to the growth medium improved growth of the organism on both citrate and D-isocitrate, suggesting that D-isocitrate was taken up by CitM in complex with divalent metal ions. Subsequently, the ability of CitM to transport D-isocitrate was demonstrated by competition experiments and by heterologous exchange in right-side-out membrane vesicles prepared from E. coli cells expressing citM. None of the other TCA cycle intermediates and related compounds tested were recognized by CitM. Uptake experiments using radioactive 63Ni2+ provided direct evidence that D-isocitrate is transported in complex with divalent metal ions.

The manufacture of yoghurt relies on the simultaneous utilization of two starters: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus). A protocooperation usually takes place between the two species, which often results in enhanced milk acidification and aroma formation compared to pure cultures. Cell-wall proteinases of Lactococcus lactis and lactobacilli have been shown to be essential to growth in milk in pure cultures. In this study, the role of proteinases PrtS from S. thermophilus and PrtB from Lb. bulgaricus in bacterial growth in milk was evaluated; a negative mutant for the prtS gene of S. thermophilus CNRZ 385 was constructed for this purpose. Pure cultures of S. thermophilus CNRZ 385 and its PrtS-negative mutant were made in milk as well as mixed cultures of S. thermophilus and Lb. bulgaricus: S. thermophilus CNRZ 385 or its PrtS-negative mutant was associated with several strains of Lb. bulgaricus, including a PrtB-negative strain. The pH and growth of bacterial populations of the resulting mixed cultures were followed, and the Lactobacillus strain was found to influence both the extent of the benefit of Lb. bulgaricus/S. thermophilus association on milk acidification and the magnitude of S. thermophilus population dominance at the end of fermentation. In all mixed cultures, the sequential growth of S. thermophilus then of Lb. bulgarius and finally of both bacteria was observed. Although proteinase PrtS was essential to S. thermophilus growth in milk in pure culture, it had no effect on bacterial growth and thus on the final pH of mixed cultures in the presence of PrtB. In contrast, proteinase PrtB was necessary for the growth of S. thermophilus, and its absence resulted in a higher final pH. From these results, a model of growth of both bacteria in mixed cultures in milk is proposed.

When mucoid (alginate-producing) Pseudomonas aeruginosa FRD1 is grown under low oxygen conditions in liquid culture (static), non-mucoid variants appear and eventually predominate. This conversion is not readily observed in aerobic, shaken cultures or static cultures containing the alternative electron acceptor nitrate. In this study, it is shown that the non-mucoid variants that arise under static growth conditions are almost exclusively algT mutants. It has been shown that AlgT not only positively regulates alginate biosynthesis, but also directly or indirectly negatively regulates flagellum synthesis. Indeed, during static growth, conversion to the non-mucoid phenotype is accompanied by the acquisition of flagellum-mediated motility. Surprisingly, by using a reporter gene fusion with the fliC promoter (pfliC::xylE), it was found that fliC expression begins within hours of static growth and is reversible after returning the culture to shaking conditions. The ability of the strain to produce alginate seems to be irrelevant to this phenomenon, as an AlgT+ ΔalgD strain showed identical results. Thus, it is suggested that the first effect of static growth is to induce motility as an adaptive measure in the presence of wild-type algT. This may afford P. aeruginosa the ability to swim towards areas of higher oxygen concentrations. Subsequent to this, algT mutations are likely to secure the motile phenotype.

Many bacteria can grow on chloroaromatic pollutants because they can transform them into chlorocatechols, which are further degraded by enzymes of a specialized ortho-cleavage pathway. Ralstonia eutropha JMP134 is able to grow on 3-chlorobenzoate by using two pJP4-encoded, ortho-cleavage chlorocatechol degradation gene clusters (tfdC I D I E I F I and tfdD II C II E II F II). Very little is known about the acquisition of new catabolic genes encoding enzymes that lead to the formation of chlorocatechols in R. eutropha JMP134. The effect on the catabolic properties of an R. eutropha JMP134 derivative that received the xylS–xylXYZL gene module, encoding the xylS-regulated expression of the broad-substrate-range toluate 1,2-dioxygenase (xylXYZ) and the 1,2-dihydro-1,2-dihydroxytoluate dehydrogenase (xylL) from pWW0, which allows the transformation of 4-chlorobenzoate into 4-chlorocatechol, was studied. Such a derivative could efficiently grow on 4-chlorobenzoate. Unexpectedly, this derivative also grew on 3,5-dichlorobenzoate, a substrate for XylXYZL but not an inducer of the XylS regulatory protein. The ability to grow on 4-chlorobenzoate or 3,5-dichlorobenzoate was also observed in derivatives of strain JMP134 containing the xyl gene module but lacking xylS, indicating the presence of an xylS-like element in R. eutropha with an inducer profile different from that of the pWW0-encoded regulator. Growth on 4-chlorobenzoate was also observed after introduction of the xyl gene module into strain JMP222, a JMP134 derivative lacking pJP4, but only if multiple copies of tfdC I D I E I F I or tfdD II C II E II F II were present. However, only the derivative containing multiple copies of tfdD II C II E II F II was able to grow on 3,5-dichlorobenzoate. These observations indicate that although the acquisition of new catabolic genes actually enhances the catabolic abilities of R. eutropha JMP134, these new properties are strongly influenced by the dosage of the tfd genes, the presence of a chromosomal xylS-like regulatory element and the different contributions of the tfd gene clusters.

The transcriptome of Bacillus subtilis was analysed at different time points (30, 60 and 90 min) after a temperature downshift from 37 to 18 °C using DNA macroarrays. This approach allowed the identification of around 50 genes exhibiting an increased mRNA level and around 50 genes exhibiting a decreased mRNA level under cold-shock conditions. Many of the repressed genes encode enzymes involved in the biosynthesis of amino acids, nucleotides and coenzymes, indicating metabolic adaptation of the cells to the decreased growth rate at the lower temperature. The strongest cold-inducible gene encodes fatty acid desaturase, which forms unsaturated fatty acids from saturated phospholipid precursors, thereby increasing membrane fluidity. The cold-shock-induced increase of mRNA levels of the classical cold-shock genes cspB, cspC and cspD could be verified. Furthermore, besides many genes encoding proteins of unknown function, some genes encoding ribosomal proteins were transcriptionally up-regulated, which points to an adaptive reprogramming of the ribosomes under cold-shock conditions. Interestingly, the amount of mRNA specified by the operon ptb-bcd-buk-lpd-bkdA1-bkdA2-bkdB, which encodes enzymes involved in degradation of branched-chain amino acids, also increases after a temperature downshift. As cells utilize the isoleucine and valine degradation intermediates α-methylbutyryl-CoA and isobutyryl-CoA for synthesis of branched-chain fatty acids, this finding reflects the adaptation of membrane lipid composition, ensuring the maintenance of appropriate membrane fluidity at low temperatures. The results of the DNA array analyses were verified for several selected genes by RNA slot-blot analysis and compared with two-dimensional PAGE analyses.

Gene pth encodes peptidyl-tRNA hydrolase (Pth), an enzyme that cleaves peptidyl-tRNAs released abortively from ribosomes during protein synthesis. In the Escherichia coli chromosome, pth is flanked by ychH and ychF, two genes of unknown function. Pth is essential for cell viability, especially under conditions leading to overproduction of peptidyl-tRNA. In an attempt to unveil the elements that affect pth expression, the transcriptional features of the pth region were investigated. Northern blot experiments showed that both pth and ychF, the 3′-proximal gene, are cotranscribed in a bicistronic transcript. However, transcripts containing each of the individual messages were also detected. Accordingly, two transcriptional promoters were identified by primer extension experiments: one located upstream of pth, which presumably gives rise to both the mono and bicistronic pth transcripts, and the other, preceding ychF, which generates its monocistronic message. Deletion analysis indicates that pth transcript stability depends on ychF integrity. Also, a defect in RNase E activity resulted in Pth overproduction. It is proposed that RNase E processing within ychF in the bicistronic message limits pth expression.

Mannitol metabolism in Lactococcus lactis MG1363 and in a derivative strain deficient in lactate dehydrogenase (LDHd) was characterized. Both strains had the ability to grow on mannitol as an energy source, although this polyol was a poorer substrate for growth than glucose. When compared to glucose, the metabolism of mannitol caused an NADH burden due to formation of an additional NADH molecule at the reaction catalysed by mannitol-1-phosphate dehydrogenase (Mtl1PDH). This resulted in a prominent accumulation of mannitol 1-phosphate (Mtl1P) both in growing and resting cells, suggesting the existence of a severe bottleneck at Mtl1PDH. Growth on mannitol induced the activity of Mtl1PDH in both the LDHd and MG1363 strains. The lower accumulation of Mtl1P in mannitol-grown cells when compared to glucose-grown LDHd cells, as monitored by in vivo 13C-NMR, reflects this induction. A clear shift towards the production of ethanol was observed on mannitol, indicating pressure to regenerate NAD+ when this substrate was used. A strategy to obtain a mannitol-overproducing strain is proposed.