- Volume 146, Issue 12, 2000

Trophozoites of the microaerophilic flagellate parasitic protozoon Giardia intestinalis have only a limited capacity to detoxify O2. Thus, when exposed to controlled concentrations of dissolved O2 >8 μM, they gradually lose their ability to scavenge O2. In a washed cell suspension stirred under 10% air in N2 (equivalent to 25 μM O2), inactivation of the O2-consuming system was complete after 3·5 h; during this period accumulation of H2O2 (3 μmol per 106 organisms) and oxidation of cellular thiols to 16% of their initial level occurred. Under 20% air (50 μM O2), respiratory inactivation was complete after 1·5 h, and under air (258 μM O2), after 50 min. Loss of O2-consuming capacity was accompanied by loss of motility. Use of the fluorogen 2,7-dichlorodihydrofluorescein acetate indicated that intracellular H2O2 is produced at extranuclear sites. Flow cytometric estimation of the plasma membrane electrochemical potentials using bis(1,3-dibutylbarbituric acid) trimethine oxonol, DiBAC4(3), showed that values declined from −134 mV to −20 mV after 4·5 h aeration. Incubation of organisms with 60 μM H2O2 for 10 min gave partial collapse of plasma membrane potential and complete loss of O2 uptake capacity; motility and viability as assessed by DiBAC4(3) exclusion were completely lost after 1 h. Inactivation of the O2-consuming system and loss of viability were also observed on exposure to singlet oxygen photochemically generated from rose bengal or toluidine blue.

Moenomycin is a natural product glycolipid that inhibits the growth of a broad spectrum of Gram-positive bacteria. In Escherichia coli, moenomycin inhibits peptidoglycan synthesis at the transglycosylation stage, causes accumulation of cell-wall intermediates, and leads to lysis and cell death. However, unlike Esc. coli, where 5–6 log units of killing are observed, 0–2 log units of killing occurred when Gram-positive bacteria were treated with similar multiples of the MIC. In addition, bulk peptidoglycan synthesis in intact Gram-positive cells was resistant to the effects of moenomycin. In contrast, synthetic disaccharides based on the moenomycin disaccharide core structure were identified that were bactericidal to Gram-positive bacteria, inhibited cell-wall synthesis in intact cells, and were active on both sensitive and vancomycin-resistant enterococci. These disaccharide analogues do not inhibit the formation of N-acetylglucosamine-β-1,4-MurNAc-pentapeptide-pyrophosphoryl-undecaprenol (lipid II), but do inhibit the polymerization of lipid II into peptidoglycan in Esc. coli. In addition, cell growth was required for bactericidal activity. The data indicate that synthetic disaccharide analogues of moenomycin inhibit cell-wall synthesis at the transglycosylation stage, and that their activity on Gram-positive bacteria differs from moenomycin due to differential targeting of the transglycosylation process. Inhibition of the transglycosylation process represents a promising approach to the design of new antibacterial agents active on drug-resistant bacteria.

A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M r of approximately 12000 as determined by SDS-PAGE. Its activity is stable over a pH range of 3·5–11 and at temperatures below 50 °C. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1:1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria.

The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium Brucella abortus. The gene was mapped on the large chromosome of B. abortus. It is flanked by one upstream and two downstream copies of the Brucella repeated sequence Bru-RS. Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport. Expression in Xenopus oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel. Glycerol uptake experiments in E. coli also showed that the protein is not able to transport glycerol.

The Bacillus subtilis aprE gene encodes subtilisin, an extracellular proteolytic enzyme produced in stationary phase. The authors examined the stability of aprE mRNA and aprE leader–lacZ fusion mRNA. Both mRNAs were found to be unusually stable, with half-lives longer than 25 min, demonstrating that the aprE leader contains a determinant for extreme mRNA stability. The half-lives were the same in growing and stationary-phase cells. This contrasts with the findings of O. Resnekov et al. (1990) R31 [Proc Natl Acad Sci USA 87, 8355–8359], which suggested a growth-phase-dependent mechanism for decay of aprE mRNA. The discrepancy is explained by the techniques used. Substitution of two bases or deletion of 25 nucleotides in the aprE leader led to a major difference in its predicted secondary structure and resulted in a fivefold reduction of the half-life of aprE mRNA. The authors also determined the half-life of amyE mRNA, which encodes α-amylase, another stationary-phase, excreted enzyme and found it to be around 5 min. This shows that extreme stability is not a general property of stationary-phase mRNAs encoding excreted enzymes.

Bacillus subtilis can utilize the purine bases adenine, hypoxanthine and xanthine as nitrogen sources. The utilization of guanine as a nitrogen source is reported here. The first step is the deamination of guanine to xanthine catalysed by guanine deaminase (GDEase). To isolate mutants defective in GDEase activity, a collection of mutant strains was screened for strains unable to use guanine as a nitrogen source. The strain BFA1819 (yknA) showed the expected phenotype and no GDEase activity could be detected in this strain. A new name for yknA, namely gde, is proposed. The gde gene encodes a 156 amino acid polypeptide and was preceded by a promoter sequence that is recognized by the σA form of RNA polymerase. High levels of GDEase were found in cells grown with purines and intermediary compounds of the purine catabolic pathway as nitrogen sources. Allantoic acid, most likely, is a low molecular mass inducer molecule. The level of GDEase was found to be subjected to global nitrogen control exerted by the GlnA/TnrA-dependent signalling pathway. The two regulatory proteins of this pathway, TnrA and GlnR, indirectly and positively affected gde expression. This is the first instance of a gene whose expression is positively regulated by GlnR. The GDEase amino acid sequence shows no homology with the mammalian enzyme. In agreement with this are the different physiological roles for the two enzymes.

Although much is known about the bacterial cellulosome and its various protein components, their contributions to bacterial growth on cellulose and the process of cellulolysis in vivo cannot currently be assessed. To remedy this, the authors have developed gene transfer techniques for Clostridium cellulolyticum ATCC 35319. Firstly, transfer of Tn1545 has been obtained using an Enterococcus faecalis donor. Secondly, IncP-mediated conjugative mobilization of plasmids from Escherichia coli donors has also been achieved. The yield of transconjugants in both cases was low and was probably limited by the suboptimal growth conditions that must of necessity be employed for the co-culture of oligotrophic C. cellulolyticum with copiotrophic donors. A restriction endonuclease was detected in crude extracts of C. cellulolyticum. This enzyme, named CceI, is an isoschizomer of MspI (HpaII). Electro-transformation was employed to establish plasmids containing the replication functions of pAMβ1 (En. faecalis), pIM13 (Bacillus subtilis), pCB102 (Clostridium butyricum), pIP404 (Clostridium perfringens) and pWV01 (Lactococcus lactis subsp. cremoris) in C. cellulolyticum. Transformants were only obtained if the DNA was appropriately methylated on the external C of the sequence 5′-CCGG-3′ using either BsuFI methylase in vivo or MspI methylase in vitro. Plasmids based on the pAMβ1 and pIM13 replicons were more stably maintained than one based on the pCB102 replicon. Selection of transformants on solid medium led to low apparent transformation efficiencies (approx. 102 transformants per μg DNA) which might, in part, reflect the low plating efficiency of the organism. Selection of transformants in liquid medium led to a higher apparent yield of transformants (between 105 and 107 transformants per μg DNA). The methods developed here will pave the way for functional analysis of the various cellulosome components in vivo.

The potential for natural genetic transformation among the seven genomovars (gvs) of Pseudomonas stutzeri was investigated. Of the 12 strains originating from a variety of environments, six strains (50%) from five gvs were competent for DNA uptake (RifR marker). The transformation frequencies varied over more than three orders of magnitude. With three highly transformable strains (ATCC 17587, ATCC 17641, JM300) from two gvs and all other strains as DNA donors, sexual isolation from other pseudomonad species (Pseudomonas alcaligenes, Pseudomonas mendocina) and also from other P. stutzeri gvs was observed (i.e. heterogamic transformation was reduced). For ATCC 17587 (gv 2) and ATCC 17641 (gv 8), heterogamic transformation was up to two and three orders of magnitude lower with other P. stutzeri gv and the other species employed, respectively, than in homogamic transformations. Interestingly, whereas with ATCC 17587 and ATCC 17641 heterogamic transformation with donors of the same gv was as high as homogamic transformation, JM300 (gv 8) was sexually isolated from its nearest relative (ATCC 17641). Also, sexual isolation of JM300 from other P. stutzeri gvs was most pronounced among the recipients tested, in some cases reaching the highest levels found with the other species as DNA donors (reduction of heterogamic transformation by 4000-fold). Results obtained here from nucleotide sequence analysis of part (422 nt) of the gene for the RNA polymerase β subunit (rpoB) from various strains indicated that sexual isolation of ATCC 17641 increased with nucleotide sequence divergence. Implications of the observed great heterogeneity in transformability, competence levels and sexual isolation among strains are discussed with regard to the evolution of P. stutzeri.

Environmental regulation of bacterial gene expression is often mediated by two-component signal transduction systems, which are themselves tightly regulated. The response regulator RegX3 and the cytoplasmic portion of the histidine kinase SenX3 from Mycobacterium bovis BCG were overproduced in Escherichia coli and purified as N-terminally (His)6-tagged proteins. Phosphorylation assays demonstrated autophosphorylation of the cytoplasmic portion of SenX3 and a phosphotransfer from SenX3 to RegX3, involving conserved histidine and aspartate residues, respectively. In addition, as shown by electrophoretic mobility shift assays, (His)6RegX3 was able to specifically bind to the promoter region of the senX3–regX3 operon. Furthermore, operon fusion analyses indicated that the overexpression of the senX3–regX3 operon increases the activity of the senX3 promoter in Mycobacterium smegmatis. Together, these results indicate that the mycobacterial SenX3–RegX3 two-component system is positively autoregulated.

The production of extracellular enzymes by Pseudomonas fluorescens is important with respect to phytopathogenesis and, in the case of psychrotrophic strains, food spoilage. The production of extracellular protease has been previously reported to be dependent on temperature in psychrotrophic strains of P. fluorescens; production is decreased above the optimum growth temperature with a relatively small change in growth rate. In this work, a transposon mutant of P. fluorescens LS107d2 has been isolated which, in contrast to the wild-type strain, is completely protease deficient at 29 °C, above the optimum growth temperature of 25 °C, but which produces protease at 23 °C. Further analysis revealed that this mutation is in a gene (prtR) which is part of a dicistronic operon, prtIR, in which the two genes are translationally coupled. Evidence is presented that prtI encodes a sigma factor related to others involved in extracytoplasmic functions (ECF sigma factors) and that prtR encodes a novel transmembrane activator of PrtI. PrtI, like PrtR, is also required for protease production at 29 °C but not at 23 °C. Analysis of the amino acid sequence of PrtR indicates that it is functionally related to a group of membrane-associated anti-sigma factors and a few transmembrane regulators, but is not significantly sequence related. Complementation analysis indicates that PrtR may also interact with sigma factors other than PrtI. The promoter region of the protease-encoding gene (aprX) in LS107d2 has been identified and has sequence features which could indicate interaction with either an ECF sigma factor or a primary sigma factor.

The yeiL open reading frame located at 48·5 min (2254 kb) in the nfo–fruA region of the Escherichia coli chromosome was predicted to encode a CRP and FNR paralogue capable of forming inter- or intra-molecular disulphide bonds and incorporating one iron–sulphur centre per 25 kDa subunit. Purified MBP–YeiL (a maltose-binding-protein–YeiL fusion protein) was a high-molecular-mass oligomer or aggregate which released unstable monomers (68 kDa) under reducing conditions. The MBP–YeiL protein contained substoichiometric amounts of iron and acid-labile sulphide, and an average of one disulphide bond per monomer. The iron and sulphide contents increased consistent with the acquisition of one [4Fe–4S] cluster per monomer after anaerobic NifS-catalysed reconstitution. By analogy with FNR and FLP (the FNR-like protein of Lactobacillus casei) it was suggested that the transcription-regulatory activity of YeiL might be modulated by a sensory iron–sulphur cluster and/or by reversible disulphide bond formation. A yeiL–lacZ transcriptional fusion showed that aerobic yeiL expression increases at least sixfold during stationary phase, requires RpoS, and is positively autoregulated by YeiL, positively activated by Lrp (and IHF in the absence of FNR) and negatively regulated by FNR. A regulatory link between the synthesis of YeiK (a potential nucleoside hydrolase) and YeiL was inferred by showing that the yeiK and yeiL genes are divergently transcribed from overlapping promoters. A 10–15% deficiency in aerobic growth yield and an enhanced loss of viability under nitrogen starvation conditions were detected with a yeiL::kan R mutant, suggesting that YeiL might function as a post-exponential-phase nitrogen-starvation regulator.

A strain that allows the cloning of Fur-regulated loci was constructed. The strain, named FUR-SEL1, contains a chromosomal fhuA′–′cat transcriptional fusion that is expressed from the Fur-regulated promoter, fhuA p. Therefore, Fur boxes introduced on a multicopy plasmid can cause derepression of the fusion by titrating the Fur repressor and thereby confer chloramphenicol resistance, which can be used as a selectable phenotype for cloning Fur-regulated loci. However, a number of additional mutations had to be introduced before FUR-SEL1 could be used for cloning Fur-regulated genes. The principal approach consisted of introducing a leaky fur mutation that ensures a more than 106-fold increase in chloramphenicol resistance for FUR-SEL1 transformants carrying FUR-box-containing plasmids. To verify that the cloning procedure selects Fur-regulated genes, 10 recombinant plasmids chosen at random among the ones selected with FUR-SEL1 were analysed by FURTA (Fur-titration assay), a method for identification of Fur-regulated genes. In addition, the nucleotide sequences of their chromosomal inserts were determined. Besides known Fur-regulated genes, seven Escherichia coli loci which have not previously been shown to be Fur regulated were found, including the pgmA and nrdHIEF genes, predicted ORF yhhY and four promoters identified first in this study. Three of the promoters preceded the nohA gene, and ORFs ygaC and yhhX. The fourth was located upstream of orf78 predicted in this work. The regulation of the promoter activities by iron and the involvement of Fur in this regulation were shown. Employing FUR-SEL1 for cloning Fur-regulated loci from other enterobacteria is discussed.

Halogenated furanones produced by the benthic marine macroalga Delisea pulchra inhibit swarming motility of Serratia liquefaciens MG1. This study demonstrates that exogenously added furanones control transcription of the quorum sensing regulated gene swrA in competition with the cognate signal molecule N-butanoyl-L-homoserine lactone. This in turn results in reduced production of the surface-active compound serrawettin W2, which is crucial for surface translocation of the differentiated swarm cells. It is demonstrated that furanones interfere with interspecies communication during swarming of mixed cultures and that the mode of interference in quorum-sensing control and interspecies communication is not through inhibition of autoinducer synthesis.

WD-repeat proteins are found in all eukaryotes and are implicated in a variety of regulatory functions as a result of protein–protein interactions. PkwA from Thermomonospora curvata CCM3352 is a first potential example of a WD-repeat protein in a prokaryotic actinomycete. A mAb (3G2) was generated against the carboxy terminus of PkwA and was used to analyse the expression of PkwA in T. curvata. PkwA was detected in exponential growth phase following inoculation with spores, but could not be found at any stage of growth following inoculation with vegetative mycelium. PkwA and its WD domain were expressed in Escherichia coli as His-tag derivatives and purified on a Talon metal affinity matrix. The WD domain was phosphorylated by Pkg2, a membrane-spanning protein Ser/Thr kinase from ‘Streptomyces granaticolor’. A membrane fraction from an exponential, spore-derived culture of T. curvata was found to phosphorylate the WD domain specifically in the presence of Mn2+. These data confirm that PkwA is expressed in spore-derived exponential growth phase of T. curvata and could play a role as a molecular switch in a signalling pathway.