- Volume 147, Issue 3, 2001

Plantaricin W (Plw) is a new two-peptide bacteriocin, from Lactobacillus plantarum, which inhibits a large number of Gram-positive bacteria. The two peptides, Plwα (comprising 29 residues) and Plwβ (comprising 32 residues), were isolated from the culture supernatants and characterized. The individual peptides had low antimicrobial activity but acted synergistically, and synergism was seen at all mixing ratios tested. The data indicate that the two peptides work in a 1:1 ratio. Chemical analyses showed that both peptides are lantibiotics, but two unmodified cysteines and one serine residue were present in Plwα, and Plwβ contained one cysteine residue. The Plw structural genes were sequenced and shown to encode prepeptides with sequence similarities to two other two-peptide lantibiotics, namely staphylococcin C55 and lacticin 3147. The conserved residues are mainly serines, threonines and cysteines that can be involved in intramolecular thioether bond formation in the C-terminal parts of the molecules. This indicates that these bacteriocins are members of a new family of lantibiotics with common bridging patterns, and that the ring structures play an important functional role. Based on the data a structural model is presented in which each peptide has a central lanthionine and two overlapping thioether bridges close to their C-termini.

Butane-grown ‘Pseudomonas butanovora’ expressed two soluble alcohol dehydrogenases (ADHs), an NAD+-dependent secondary ADH and an NAD+-independent primary ADH. Two additional NAD+-dependent secondary ADHs could be detected when cells were grown on 2-butanol and lactate. The inducible NAD+-independent 1-butanol dehydrogenase (BDH) of butane-grown cells was primarily responsible for 1-butanol oxidation in the butane metabolism pathway. BDH was purified to near homogeneity and identified as a quinohaemoprotein, containing, per mol enzyme, 1·0 mol pyrroloquinoline quinone (PQQ) and 0·25 mol haem c as prosthetic groups. BDH was synthesized as a monomer of approximately 66 kDa. It has a broad substrate range, including primary alcohols, secondary alcohols, aldehydes, C4 diols and aromatic alcohols. It exhibited the lowest K m (7±1 μM) and highest k cat/K m (72×104 M−1 s−1) value towards 1-butanol. BDH exhibited ferricyanide-dependent ADH activity. Calcium ions (up to 10 mM) increased BDH activity substantially. Two BDH internal amino acid sequences showed 73 and 62% identity and 83 and 66% similarity, respectively, when compared with an amino acid sequence of ethanol dehydrogenase from Comamonas testosteroni. The presence of the inducible BDH and secondary ADH may indicate that the terminal and subterminal oxidation pathways are involved in butane degradation of butane-grown ‘P. butanovora’.

The phage-display cloning technique was used to find rhizobial proteins that bind to receptors located on the bacterial cell surface. The aim was to clone the gene(s) encoding rhicadhesin, a universal rhizobial adhesion protein, and/or other cell-surface-binding proteins. Four such R hizobium-adhering proteins (Rap) were revealed in Rhizobium leguminosarum bv. trifolii strain R200. The binding is mediated by homologous Ra domains in these proteins. One member of the Rap protein family, named RapA1, is a secreted calcium-binding protein, which are also properties expected for rhicadhesin. However, the size of the protein (24 kDa instead of 14 kDa) and its distribution among different rhizobia (present in only Rhizobium leguminosarum biovars and R. etli instead of all members of Rhizobiaceae) argue against RapA1 being rhicadhesin. Protein RapA1 consists of two homologous Ra domains and agglutinates R200 cells by binding to specific receptors located at one cell pole during exponential growth. Expression of these cell-surface receptors was detected only in rhizobia that produce the RapA proteins. The authors propose that the homologous Ra domains, found to be present also in other proteins with different structure, represent lectin domains, which confer upon these proteins the ability to recognize their cognate carbohydrate structures.

Flavobacterium heparinum (now Pedobacter heparinus) is a Gram-negative soil bacterium which can produce yellow pigments. It synthesizes five enzymes that degrade glycosoaminoglycan molecules. The study of this unique bacterium has been limited by the absence of a genetic manipulation system. In this paper, the construction of a conjugation/integration plasmid system and a broad-host-range plasmid, both of which contain a F. heparinum functional selective marker created by placing the trimethoprim resistance gene, dhfrII, under the control of the hepA regulatory region is described. Both plasmids were introduced into F. heparinum by conjugation and/or electroporation, and trimethoprim resistant colonies were obtained. Fifty electroporants were obtained per microgram covalently closed circular plasmid DNA. The existence of integrated plasmid DNA was confirmed by Southern hybridization and PCR. The existence of a derivative of the broad-host-range plasmid pBBR1 in F. heparinum was demonstrated by plasmid digestion and Southern hybridization, and by transformation of Escherichia coli.

Carbohydrate-binding modules (CBMs) are often part of the complex hydrolytic extracellular enzymes from bacteria and may modulate their catalytic activity. The thermostable catalytic domain of laminarinase Lam16A from Thermotoga neapolitana (glycosyl hydrolase family 16) is flanked by two CBMs, 148 and 161 aa long. They share a sequence identity of 30%, are homologous to family CBM4 and are thus called CBM4-1 and CBM4-2 respectively. Recombinant Lam16A proteins deleted for one or both binding modules and the isolated module CBM4-1 were characterized. Proteins containing the N-terminal module CBM4-1 bound to the soluble polysaccharides laminarin (1,3-β-glucan) and barley 1,3/1,4-β-glucan, and proteins containing the C-terminal module CBM4-2 bound additionally to curdlan (1,3-β-glucan) and pustulan (1,6-β-glucan), and to insoluble yeast cell wall β-glucan. The activity of the catalytic domain on soluble 1,3-β-glucans was stimulated by the presence of CBM4-1, whereas the presence of CBM4-2 enhanced the Lam16A activity towards gelatinized and insoluble or mixed-linkage 1,3-β-glucan. Thermostability of the catalytic domain was not affected by the truncations. Members of family CBM4 can be divided into four subfamilies, members of which show different polysaccharide-binding specificities corresponding to the catalytic specificities of the associated hydrolytic domains.

Albicidins, a family of potent antibiotics and phytotoxins produced by the sugarcane leaf scald pathogen Xanthomonas albilineans, inhibit DNA replication in bacteria and plastids. A gene located by Tn5-tagging was confirmed by complementation to participate in albicidin biosynthesis. The gene (xabB) encodes a large protein (predicted M r 525695), with a modular architecture indicative of a multifunctional polyketide synthase (PKS) linked to a non-ribosomal peptide synthetase (NRPS). At 4801 amino acids in length, XabB is the largest reported PKS–NRPS. Twelve catalytic domains in this multifunctional enzyme are arranged in the order N terminus–acyl-CoA ligase (AL)–acyl carrier protein (ACP)–β-ketoacyl synthase (KS)–β-ketoacyl reductase (KR)–ACP–ACP–KS–peptidyl carrier protein (PCP)–condensation (C)–adenylation–PCP–C. The modular architecture of XabB indicates likely steps in albicidin biosynthesis and approaches to enhance antibiotic yield. The novel pattern of domains, in comparison with known PKS–NRPS enzymes for antibiotic production, also contributes to the knowledge base for rational design of enzymes producing novel antibiotics.

The role of the Lactobacillus pentosus phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) in sugar transport and control of sugar utilization was investigated. Growth experiments and measurements of PEP-dependent phosphorylation of sugars, of sugar transport and of catabolic enzyme activity were performed, to compare a wild-type strain with an EIIBMan mutant, LPE6, and a ccpA mutant, LPE4. Fructose uptake in wild-type bacteria demonstrated the presence of two fructose-specific PTSs: a high-affinity system, EIIFru (K m=52 μM) which is inducible by fructose, and a low-affinity system (K m=300 μM). The latter system was lacking in LPE6 and therefore corresponds to EIIMan. LPE6 was unable to phosphorylate glucose, mannose, N-acetylglucosamine and 2-deoxyglucose in a PEP-dependent reaction, indicating that these sugars are substrates of EIIMan. Transport and phosphorylation of these compounds was the same in LPE4 and in wild-type bacteria, although growth of LPE4 on these sugars was impaired. In wild-type bacteria and in LPE4 the activity of EIIFru was lowered by the presence of EIIMan substrates in the growth medium, but this decrease was not observed in LPE6. These results indicate that EIIMan but not CcpA regulates the synthesis of EIIFru. Mutations in EIIMan or CcpA resulted in a relief of catabolite repression exerted by EIIMan substrates on the activity of β-galactosidase and β-glucosidase, indicating that EIIMan and CcpA are important components in catabolite repression in L. pentosus. Fructose-mediated repression of these two enzymes appeared to be correlated with the activity of EIIFru.

The rplK gene of Corynebacterium glutamicum ATCC13032 comprises 438 nucleotides and encodes a protein of 145 amino acids with a molecular mass of 15·3 kDa. The amino acid sequence revealed extensive similarities to the large ribosomal subunit protein L11 from several Gram-positive and Gram-negative bacteria. The C. glutamicum rplK gene is located downstream of secE, representing part of the protein export apparatus, and of nusG, encoding a transcription antiterminator protein. The rplK gene is followed by an ORF homologous to rplA encoding the 50S ribosomal protein L1. Northern analysis revealed that transcription of the rplK–rplA cluster resulted in two different transcripts of 1·5 and 0·6 kb. The 1·5 kb transcript corresponds to the entire rplK–rplA cluster and the short transcript originates from the rplK gene. A C. glutamicum rplK mutant strain carrying a 12 bp in-frame deletion within rplK, which resulted in the loss of the tetrapeptide Pro-Ala-Leu-Gly in the L11 protein, was constructed. The mutant failed to accumulate (p)ppGpp in response to amino acid starvation and exhibited an increased tolerance to the antibiotic thiostrepton. Evidently, the C. glutamicum rplK gene is required for (p)ppGpp accumulation upon nutritional starvation.

Salmonella typhimurium ATCC14028 readily multiplies in professional phagocytes in vitro and is highly virulent in mice. Mutants lacking thymidylate synthase activity (thyA) were isolated and shown to be strictly dependent on thymidine monophosphate precursors in the growth medium. The thyA mutants were found to be virtually incapable of intracellular growth and survival in vitro, both in macrophage-like cell line P338D1 and in the human epithelial cell line Hep-2, and their virulence was impaired in BALB/c mice. Intraperitoneal immunization of mice with two doses of live S. typhimurium thyA provided protection against a challenge with 103 times the 50% lethal dose of the virulent parent strain.

Campylobacter upsaliensis is an emerging human enteropathogen. However, little is known about the pathogenesis of C. upsaliensis infection. In this study the authors demonstrate that C. upsaliensis whole-cell preparations and extracts produce a cytolethal distending toxin (CDT)-like effect on HeLa cells characterized by progressive distension and nuclear fragmentation culminating in cell death over 5 d. To further delineate the nature of this toxic effect in relation to CDT from other pathogens, the effect of C. upsaliensis on cellular events in epithelial cells and immunocytes was investigated. C. upsaliensis lysate-treated HeLa cells subjected to FACScan analysis using carboxyfluorescein diacetete succinimidyl ester (CFDA-SE) as a cell tracer demonstrated cell division arrest. Propidium iodide (PI) staining of HeLa cells revealed that cell cycle arrest occurred in G2/M. Human T lymphocytes exposed to C. upsaliensis lysates also showed cell cycle arrest in G2/M. Using a combination of Annexin V/PI staining and TUNEL assay, cytodistended HeLa cells were shown to undergo apoptotic cell death. These data provide the first insights into the virulence mechanisms of this novel enteropathogen.

A copper-transport (copYAZ) operon was cloned from the oral bacterium Streptococcus mutans JH1005. DNA sequencing showed that the operon contained three genes (copY, copA and copZ), which were flanked by a single promoter and a factor-independent terminator. copY encoded a small protein of 147 aa with a heavy-metal-binding motif (CXCX4CXC) at the C-terminus. CopY shared extensive homology with other bacterial negative transcriptional regulators. copA encoded a 742 aa protein that shared extensive homology with P-type ATPases. copZ encoded a 67 aa protein that also contained a heavy-metal-binding motif (CXXC) at the N-terminus. Northern blotting showed that a 3·2 kb transcript was produced by Cu2+-induced Strep. mutans cells, suggesting that the genes were synthesized as a polycistronic message. The transcriptional start site of the cop operon was mapped and shown to lie within the inverted repeats of the promoter–operator region. Strep. mutans wild-type cells were resistant to 800 μM Cu2+, whereas cells of a cop knock-out mutant were killed by 200 μM Cu2+. Complementation of the cop knock-out mutant with the cop operon restored Cu2+ resistance to wild-type level. The wild-type and the mutant did not show any differences in susceptibility to other heavy metals, suggesting that the operon was specific for copper. By using a chloramphenicol acetyltransferase reporter gene fusion, the cop operon was shown to be negatively regulated by CopY and could be derepressed by Cu2+.