- Volume 147, Issue 12, 2001

Expression of the UhpT sugar-phosphate transporter in Escherichia coli is regulated at the transcriptional level via the UhpABC signalling cascade. Sensing of extracellular glucose 6-phosphate (G6P), by membrane-bound UhpC, modulates a second membrane-bound protein, UhpB, resulting in autophosphorylation of a conserved histidine residue in the cytoplasmic (transmitter) domain of the latter. Subsequently, this phosphoryl group is transferred to a conserved aspartate residue in the response-regulator UhpA, which then initiates uhpT transcription, via binding to the uhpT promoter region. This study demonstrates the hypothesized transmembrane signal transfer in an ISO membrane set-up, i.e. in a suspension of UhpBC-enriched membrane vesicles, UhpB autophosphorylation is stimulated, in the presence of [γ-32P]ATP, upon intra-vesicular sensing of G6P by UhpC. Subsequently, upon addition of UhpA, very rapid and transient UhpA phosphorylation takes place. When P∼UhpA is added to G6P-induced UhpBC-enriched membrane vesicles, rapid UhpA dephosphorylation occurs. So, in the G6P-activated state, UhpB phosphatase activity dominates over kinase activity, even in the presence of saturating amounts of G6P. This may imply that maximal in vivo P∼UhpA levels are low and/or that, to keep sufficient P∼UhpA accumulated to induce uhpT transcription, the uhpT promoter DNA itself is involved in stabilization/sequestration of P∼UhpA.

Genes placed under the control of the arabinose-inducible araBAD promoter (PBAD) of Escherichia coli are expressed in an all-or-none fashion, in which the percentage of induced cells in the population, rather than the degree of induction in individual cells, varies with the concentration of arabinose in the culture medium. Previous work showed that all-or-none gene expression from PBAD was due to the arabinose-dependent expression of the gene encoding the low-affinity high-capacity transporter (araE), and that expression of heterologous genes from PBAD in individual cells could be regulated by placing the araE gene under control of an arabinose-independent promoter. Based on these results, two expression systems were developed to allow regulatable control of genes under control of PBAD. In one system, the native araE promoter on the chromosome was replaced by constitutive promoters of different strengths. In the second system, the araE gene under control of the same constitutive promoters was placed on a medium-copy plasmid. Both systems allow regulatable expression of a plasmid-borne PBAD-controlled heterologous gene and a homogeneous population of cells over a wide range of arabinose concentrations. While the degree of induction varied slightly with the strength of the constitutive promoter, expression was affected most by the arabinose concentration.

Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue, novel Gfp-based biosensors for non-destructive, in situ detection of AHLs were constructed and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa, while the latter strain did not respond to the molecules produced by B. cepacia. Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

Sensitivity of Listeria monocytogenes to the bacteriocin mesentericin Y105 was previously shown to be dependent on the σ54 subunit of the RNA polymerase. This points towards expression of particular σ54-dependent genes. The present study describes first, ManR, a new σ54-associated activator, and second, \(EII_{t}^{Man}\) , a new σ54-dependent PTS permease of the mannose family, both involved in sensitivity to mesentericin Y105, since interruption of their corresponding genes led to resistance of L. monocytogenes EGDe. \(EII_{t}^{Man}\) is likely composed of three subunits encoded by the mpt operon (mptA, mptC and mptD genes). Interruption of either the proximal (mptA) or distal (mptD) gene led to resistance, supporting results obtained in Enterococcus faecalis. Accordingly, such PTS permeases of the mannose family should be involved in sensitivity of different target strains to mesentericin Y105. In L. monocytogenes, expression of the mpt operon is shown to be controlled by σ54 and ManR and to be induced by both glucose and mannose. The latter result indicates that these sugars are transported by the \(EII_{t}^{Man}\) permease. Moreover, these sugars correlatively induce sensitivity of L. monocytogenes to mesentericin Y105, strongly favouring the primary role of \(EII_{t}^{Man}\) . MptD, a membrane subunit of \(EII_{t}^{Man}\) , presents an additional domain compared to most IIDMan subunits described in data banks. An in-frame deletion of this domain in mptD led to resistance of L. monocytogenes, showing its connection with sensitivity and suggesting that it could be directly involved in the recognition of the target cell by mesentericin Y105. Taken together, the results of this work demonstrate that \(EII_{t}^{Man}\) is prominent in sensitivity to mesentericin Y105 and could be a receptor for subclass IIa bacteriocins.

In mycobacteria, as in most bacterial species, the expression of RecA is induced by DNA damage. However, the authors show here that the kinetics of recA induction in Mycobacterium smegmatis and in Mycobacterium tuberculosis are quite different: whilst maximum expression in M. smegmatis occurred 3–6 h after addition of a DNA-damaging agent, incubation for 18–36 h was required to reach peak levels in M. tuberculosis. This is despite the fact that the M. tuberculosis promoter can be activated more rapidly when transferred to M. smegmatis. In addition, it is demonstrated that in both species the DNA is sufficiently damaged to give maximum induction within the first hour of incubation with mitomycin C. The difference in the induction kinetics of recA between the two species was mirrored by a difference in the levels of DNA-binding-competent LexA following DNA damage. A decrease in the ability of LexA to bind to the SOS box was readily detected by 2 h in M. smegmatis, whilst a decrease was not apparent until 18–24 h in M. tuberculosis and then only a very small decrease was observed.

Disruption of the adhC gene of Mycobacterium smegmatis mc2155, by standard gene replacement methods, revealed that there are two copies of this gene within a large duplication of the M. smegmatis mc2155 genome. M. smegmatis AdhC+/− and M. smegmatis AdhC−/− mutants were obtained when one or two adhC copies, respectively, were disrupted by homologous recombination. Southern blot analysis of DraI restriction digests of the DNA from these mutants and from wild-type M. smegmatis mc2155, resolved by PFGE, showed that the duplication size may be at least ∼250 kb. The single and double knockout mutants were characterized and compared with the M. smegmatis wild-type. A growth disadvantage and a different morphology were associated with the loss of expression of one or both of the adhC copies, but both mutants were still acid-fast. Findings in this study indicate that the process of chromosomal duplication in M. smegmatis is ongoing and remains a potent source of genome dynamics. Hence, the M. smegmatis mc2155 genome might be larger than previously thought.

Histatins are a structurally related family of salivary proteins known as histidine-rich proteins that are produced and secreted by the human major salivary glands. In vitro, histatins are potent cytotoxic proteins with selectivity for pathogenic yeasts including Candida albicans. Studies that investigate the mechanism of action of histatin proteins upon this important human pathogen have used a candidacidal assay in which the histatin is applied extracellularly. In order to develop a model system to study the mechanism of histatin action independently from binding and translocation events, the authors constructed C. albicans strains that contain chromosomally encoded human salivary histatin genes under the control of a regulated promoter. Intracellular expression of either histatin 5 or histatin 3 induced cell killing and ATP release in parallel. Since histatin killing can be initiated solely from intracellular sites, extracellular binding and internalization are preceding transport events. Thus the mechanism of histatin-induced ATP release does not require extracellular binding, and intracellular targets alone can activate ATP release. By employing a codon-optimization strategy it was shown that expression of heterologous sequences in C. albicans can be a useful tool for functional studies.

Caulobacter crescentus was investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Polyhydroxyalkanoate (PHA) accumulation contributing to approximately 18% of the cell dry weight was obtained in the presence of glucose. Gas chromatography–mass spectrometry and gel permeation chromatography of the purified PHA showed that this polyester was solely composed of 3-hydroxybutyrate and had a weight average molar mass of 5·5×105 g mol−1 and a polydispersity of 1·6. An ORF encoding a conserved, hypothetical protein which shared approximately 47% identity with the PHB synthase from Azorhizobium caulinodans was identified within the complete C. crescentus genomic sequence. This putative C. crescentus PHB synthase gene, phaC, consisted of a 2019 nt stretch of DNA (encoding 673 aa residues), which encoded a PHB synthase with a molecular mass of approximately 73 kDa. This is currently the largest PHA synthase identified. The phaC coding region was subcloned into vector pBBR1-JO2 under lac promoter control. The resulting plasmid, pQQ4, mediated PHB accumulation in the mutant Ralstonia eutropha PHB−4 and recombinant Escherichia coli JM109(pBHR69), which produced the β-ketothiolase and acetoacetyl-CoA reductase from R. eutropha, contributing to approximately 62% and 6% of cell dry weight, respectively. Functional expression of the coding region of phaC was confirmed by immunoblotting and in vitro PHB synthase activity.

Replication of the streptococcal plasmid pIP501 is regulated by two components, CopR and the antisense RNA, RNAIII. CopR represses transcription of the essential repR mRNA about 10- to 20-fold and, additionally, prevents convergent transcription of sense and antisense RNAs. It has been demonstrated that CopR binds as a preformed dimer. DNA binding and dimerization constants were determined and amino acids were identified that are involved in DNA binding and dimerization. It was demonstrated that the C-terminal 20 aa of CopR are not involved in either activity, but play an important role for CopR stability. Furthermore, it was found that the C terminus of CopR is structured containing a β-strand structure, most probably between the alternating hydrophilic and hydrophobic amino acids 76 and 84 (QVTLELEME). In this study stability motifs within the C terminus of CopR were dissected. Both the cognate and a heterologous (QVTVTVTVT) β-strand structure between amino acids 76 and 84 within the C terminus stabilized CopR (CopR derivative CopVT). In contrast, substitution by a predicted α-helix (QVTLKLKMK) or a predicted unstructured sequence (QVTPEPEPE) caused severe and moderate destabilization, respectively. E80 seemed to be the only important C-terminal glutamic acid residue. Deletion of seven C-terminal amino acids from either wild-type CopR or CopVT reduced the half-life to ∼50% indicating that this C-terminal sequence is a second stability motif.

Silver compounds are used as antimicrobial agents in medicine and bacteria that develop resistance to silver cations (Ag+) pose problems similar to those of antibiotic-resistant bacteria. The first set of Ag+ resistance genes (sil) was from plasmid pMG101, now assigned to the IncHI incompatibility group. Questions of whether sil genes are unique to pMG101 or are more widely found, and whether they are associated with a specific incompatibility group or occur in many plasmid groups and on bacterial chromosomes were addressed. sil genes were identified in five IncH plasmids, but not in plasmids of the IncP incompatibility group. Three sil genes (silP, silR and silE) from these plasmids were PCR-amplified, cloned, sequenced and compared to those of pMG101. Differences of 0–50 nt per kb of sequence were found. Predicted gene products were 0–6% different in amino acid sequence, but the differences did not alter residues thought to be involved in protein function (see supplementary data at http://mic.sgmjournals.org or http://www.uic.edu/depts/mcmi/individual/gupta/index.htm). For representative IncH plasmid R476b and pMG101 the effects of Ag+ exposure on resistance levels were measured by growth. The inducibility of silC, silR and silE gene expression after Ag+ exposure was studied by reverse transcriptase (RT)-PCR. Silver resistance increased after Ag+ exposure for strains carrying plasmid R476b. silC and silE expression from R476b was inducible after Ag+ exposure and was constitutive and high from pMG101. The mRNA levels for the regulatory gene silR was constitutive for both pMG101 and R476b. Close homologues for silABC(ORF96)RS from pMG101 are clustered on the chromosomes of Escherichia coli strains K-12 and O157:H7, without contiguous silP and silE homologues. Insertion deletions of the E. coli K-12 chromosomal homologues for silA and silP gave Ag+ hypersensitivity for growth. The silA homologue knockout was complemented back to wild-type resistance by the same gene cloned on a plasmid. Homologues of sil genes have also been identified on other enterobacterial genomes.

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), α and β, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSHβ (cbsHβ), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsHβ, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSHα. A phenotypic screen for BSH activity and a genetic screen for the cbsHβ locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsHβ were from human sources. Variability in the BSH phenotype and cbsHβ genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsHβ locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbsβ genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

Transcription of sacB, yveB and yveA, three clustered genes on the Bacillus subtilis chromosome, is simultaneously induced by sucrose. Northern blotting analyses with specific probes showed three distinct mRNAs: a monocistronic 1·7 kb sacB mRNA, a bicistronic 3·3 kb sacB–yveB mRNA and a tricistronic 4·9 kb sacB–yveB–yveA mRNA. These results indicate that sacB, encoding levansucrase, is the proximal gene of a sucrose-inducible operon that includes the two other genes. The yield of the full-length transcript is lower than that of the bicistronic transcript, whose yield is itself lower than that of the monocistronic transcript. This suggested that the 3′ terminal parts of sacB and yveB genes worked as internal terminator structures. The protein encoded by yveB, which remains anchored to the membrane, displays an endolevanase activity, which, coupled with exolevanase activity of SacB, leads to a complete degradation of levan, a branched fructosyl polymer. It is proposed to rename yveB as levB.

Giardia intestinalis and Hexamita inflata are microaerophilic protozoa which rely on fermentative metabolism for energy generation. These organisms have developed a number of antioxidant defence strategies to cope with elevated O2 tensions which are inimical to survival. In this study, the ability of pyruvate, a central component of their energy metabolism, to act as a physiological antioxidant was investigated. The intracellular pools of 2-oxo acids in G. intestinalis were determined by HPLC. With the aid of a dichlorodihydrofluorescein diacetate-based assay, intracellular reactive oxygen species generation by G. intestinalis and H. inflata suspensions was monitored on-line. Addition of physiologically relevant concentrations of pyruvate to G. intestinalis and H. inflata cell suspensions was shown to attenuate the rate of H2O2- and menadione-induced generation of reactive oxygen species. In addition, pyruvate was also shown to decrease the generation of low-level chemiluminescence arising from the oxygenation of anaerobic suspensions of H. inflata. In contrast, addition of pyruvate to suspensions of respiring Saccharomyces cerevisiae was shown to increase the generation of reactive oxygen species. These data suggest that (i) in G. intestinalis and H. inflata, pyruvate exerts antioxidant activity at physiological levels, and (ii) it is the absence of a respiratory chain in the diplomonads which facilitates the observed antioxidant activity.