Effect of starch and amylase on the expression of amylase-binding protein A in Streptococcus gordonii.

Streptococcus gordonii is a common oral commensal bacterial species in tooth biofilm (dental plaque) and specifically binds to salivary amylase through the surface exposed amylase-binding protein A (AbpA). When S. gordonii cells are pretreated with amylase, amylase bound to AbpA facilitates growth with starch as a primary nutrition source. The goal of this study was to explore possible regulatory effects of starch, starch metabolites and amylase on the expression of S. gordonii AbpA. An amylase ligand-binding assay was used to assess the expression of AbpA in culture supernatants and on bacterial cells from S. gordonii grown in defined medium supplemented with 1% starch, 0.5 mg ml(-1) amylase, with starch and amylase together, or with various linear malto-oligosaccharides. Transcription of abpA was determined by reverse transcription quantitative polymerase chain reaction. AbpA was not detectable in culture supernatants containing either starch alone or amylase alone. In contrast, the amount of AbpA was notably increased when starch and amylase were both present in the medium. The expression of abpA was significantly increased (P < 0.05) following 40 min of incubation in defined medium supplemented with starch and amylase. Similar results were obtained in the presence of maltose and other short-chain malto-oligosacchrides. These results suggest that the products of starch hydrolysis produced from the action of salivary α-amylase, particularly maltose and maltotriose, up-regulate AbpA expression in S. gordonii.

Behavioral evidence for a glucose polymer taste receptor that is independent of the T1R2+3 heterodimer in a mouse model.

Although it is clear that the heterodimer formed by the T1R2 and T1R3 proteins serves as the primary taste receptor for sweeteners, there is growing evidence that responses to glucose polymers may be mediated by a different taste receptor. Here we report that although T1R2 knock-out (KO) and T1R3 KO mice displayed severely impaired responding to glucose, maltose, and maltotriose in an initial session of a brief-access taste test (5 s trials, 25 min sessions) relative to wild-type (WT) mice, they subsequently increased their licking as a function of concentration for maltose and maltotriose with continued testing, presumably due to associating weak oral cues with positive post-ingestive consequences. Interestingly, these KO mice displayed relatively normal concentration-dependent licking to Polycose, a mixture of glucose polymers, even in the first session. Importantly, the experience-dependent increase in responsiveness to the sugars observed with the T1R2 and T1R3 single KO mice was not statistically significant in the T1R2/3 double KO mice. The double KO mice, however, still displayed significant concentration-dependent responding to Polycose in the first test session, albeit lick rates were slightly lower than those seen for WT mice, perhaps because small amounts of glucose, maltose, and maltotriose found in Polycose were enhancing the signal in WT mice or because T1R2 or T1R3 can possibly heteromerize with another protein to form a fully functional glucose polymer receptor. These findings provide behavioral evidence that glucose polymers, with an optimal chain length greater than three glucose moieties, stimulate a taste receptor independent of the T1R2+3 heterodimer.

Molecular cloning and sequence analysis of lamB encoding outer membrane maltose-inducible porin of Aeromonas hydrophila.

Aeromonas hydrophila is a significantly important pathogen causing major diseases in humans and fresh water fish. The outer membrane proteins (OMP) which are strong immunogens have been reported to act as adhesins aiding in the attachment of enteropathogenic bacteria. It is of interest to investigate the role of OMP in pathogenesis and their potential as vaccine candidates. In our laboratory, we cloned the gene encoding channel protein LamB porin of A. hydrophila. DNA sequence analysis revealed a full length gene of 1345 bp having a high level of homology with the lamB gene of different bacteria. Open reading frame of A. hydrophila lamB consists of a signal peptide of 25 amino acids, two protein translation start sites ATG present at the 31st and 37th base pairs, a translation termination codon, TAA at 1333rd base pair.

Construction and evaluation of a chromosomal expression platform (CEP) for ectopic, maltose-driven gene expression in Streptococcus pneumoniae.

In this paper, the construction and evaluation of a chromosomal expression platform (CEP), which allows controlled gene expression following ectopic integration into the chromosome of Streptococcus pneumoniae, is described. CEP is based on the well-studied maltosaccharide-inducible system. To facilitate integration at CEP, a plasmid, pCEP, capable of replication in Escherichia coli, but not in S. pneumoniae, was assembled. This plasmid contains an expression/selection cassette flanked on each side by more than 2 kb of pneumococcal DNA. The cassette comprises a maltose-inducible promoter, P(M), separated from a kanamycin-resistance gene by NcoI and BamHI cloning sites. Clones harbouring the gene of interest integrated at CEP under the control of P(M) can be obtained through direct transformation of an S. pneumoniae recipient with ligation products between that gene and NcoI/BamHI-digested pCEP DNA, followed by selection for kanamycin-resistant transformants.

Analysis of the effect exerted by extracellular pH on the maltose regulon in Escherichia coli K-12.

The Escherichia coli maltose regulon consists of five operons under the control of the MalT transcriptional activator. lac operon fusions were constructed in vitro with the MalT-dependent promoter and with the malT promoter itself. beta-Galactosidase activity displayed by these fusions during growth at different external pH (pHo) revealed that growth at a pHo higher than 6 stimulates the transcription of malT- and MalT-controlled genes in the absence or presence of maltose. Using a malTp1 malTp10 promoter that is cAMP-CRP (cAMP receptor protein)-independent, it was demonstrated that CRP is essential for malT pHo regulation and that the pHo-dependent activity of malKp is a direct consequence of malT regulation. The pHo regulation displayed by a deleted but still functional malT promoter fused to lacZ demonstrates that this minimal promoter contains all the regulatory regions for establishing pHo regulation. In the absence of MIc, a repressor of malT expression, the pHo regulation of malT was still effective. It is proposed that binding of cAMP-CRP at malTp may be affected by malTp topology induced by pHo or that a pHo-dependent effector may act in concert with the cAMP-CRP complex.

Coding the sweet taste in the nucleus of the solitary tract: differential roles for anterior tongue and nasoincisor duct gustatory receptors in the rat.

1. A variety of chemicals that humans describe as sweet drive neurons in the nucleus of the solitary tract (NST) of the rat more vigorously when applied to the taste receptors associated with the nasoincisor ducts (NID) than when applied to taste receptors on the anterior tongue (AT). 2. The differential effects of sweet stimuli applied to the AT and NID also are evident in the set of across-neuron correlations produced by these stimuli. The psychophysical similarity among the sweet stimuli is better accounted for by responses to stimulation of the NID than by responses to stimulation of the AT (mean correlation between pairs of sweet stimuli = +0.70 for the NID, +0.44 for the AT). 3. Disaccharides or polysaccharides of glucose, i.e., maltose (0.3 M) and Polycose (0.1 M), are poor stimuli on the NID, evoking responses only 17.8 and 26.7% as great as the response elicited by sucrose (0.3 M), an optimal stimulus for this receptor subpopulation. This suggests that Polycose and maltose interact with receptor sites distinct from those with an affinity for sweet stimuli. Polycose and maltose also are ineffective stimuli on the AT, evoking responses only 11.8 and 4.9% as large as the response evoked by an optimal stimulus for this receptor subpopulation, a mixture of electrolytes (0.3 M NaCl, 0.03 M HCl, and 0.01 M quinine HCl). 4. The relative effectiveness of the sweet sugars in driving NST neurons (sucrose greater than fructose greater than glucose) correlates with their order of effectiveness in generating preference behavior in the rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartate taxis mutants of the Escherichia coli tar chemoreceptor.

The Tar protein of Escherichia coli belongs to a family of methyl-accepting inner membrane proteins that mediate chemotactic responses to a variety of compounds. These transmembrane signalers monitor the chemical environment by means of specific ligand-binding sites arrayed on the periplasmic side of the membrane, and in turn control cytoplasmic signals that modulate the flagellar rotational machinery. The periplasmic receptor domain of Tar senses two quite different chemoeffectors, aspartate and maltose. Aspartate is detected through direct binding to Tar molecules, whereas maltose is detected indirectly when complexed with the periplasmic maltose-binding protein. Saturating levels of either aspartate or maltose do not block behavioral responses to the other compound, indicating that the detection sites for these two attractants are not identical. We initiated structure-function studies of these chemoreceptor sites by isolating tar mutants which eliminate aspartate or maltose taxis, while retaining the ability to respond to the other chemoeffector. Mutants with greatly reduced aspartate taxis are described and characterized in this report. When present in single copy in the chromosome, these tar mutations generally eliminated chemotactic responses to aspartate and structurally related compounds, such as glutamate and methionine. Residual responses to these compounds were shifted to higher concentrations, indicating a reduced affinity of the aspartate-binding site in the mutant receptors. Maltose responses in the mutants ranged from 10 to 80% of normal, but had no detectable threshold shifts, indicating that these receptor alterations may have little effect on maltose detection sensitivity. The mutational changes in 17 mutants were determined by DNA sequence analysis. Each mutant exhibited a single amino acid replacement at residue 64, 69, or 73 in the Tar molecule. The wild-type Tar transducer contains arginines at all three of these positions, implying that electrostatic forces may play an important role in aspartate detection.

Hybrid Escherichia coli sensory transducers with altered stimulus detection and signaling properties.

The tar and tap loci of Escherichia coli encode methyl-accepting inner membrane proteins that mediate chemotactic responses to aspartate and maltose or to dipeptides. These genes lie adjacent to each other in the same orientation on the chromosome and have extensive sequence homology throughout the C-terminal portions of their coding regions. Many spontaneous deletions in the tar-tap region appear to be generated by recombination between these regions of homology, leading to gene fusions that produce hybrid transducer molecules in which the N terminus of Tar is joined to the C terminus of Tap. The properties of two such hybrids are described in this report. Although Tar and Tap molecules have homologous domain structures, these Tar-Tap hybrids exhibited defects in stimulus detection and flagellar signaling. Both hybrid transducers retained Tar receptor specificity, but had reduced detection sensitivity. This defect was correlated with the presence of the C-terminal methyl-accepting segment of Tap, which may have more methylation sites than its Tar counterpart, leading to elevated steady-state methylation levels in the hybrid molecules. One of the hybrids, which carried a more extensive segment from Tap, appeared to generate constitutive signals that locked the flagellar motors in a counterclockwise rotational mode. Changes in the methylation state of this transducer were ineffective in cancelling this aberrant signal. These findings implicate the conserved C-terminal domain of bacterial transducers in the generation or regulation of flagellar signals.

The use of gene fusions of study bacterial transport proteins.

The transport of solutes by bacteria has been studied for about thirty years. Early experiments on amino acid entry and galactoside accumulation provided concrete evidence that bacteria possessed specific transport systems and that these were subject to regulation. Since than a large number of transport systems have been discovered and studied extensively. Many of these use entirely different strategies for capturing or accumulating substrates. This diversity reflects variation in the availability of nutrients an ions in the different environments tolerated and inhabited by microorganisms. Examination of a few bacterial transport systems provides an opportunity to gain insight into a wide range of topics in the area of membrane transport. These include: the identification of carrier proteins and their arrangement in the membrane, the regulation of transport protein synthesis by environmental factors, and the localization of transport proteins to their extracytoplasmic destinations. It has been possible to construct a number of bacterial strains in which the gene (lacZ) which codes for the cytoplasmic enzyme beta-galactosidase is fused to genes which code for transport proteins. The following article is intended to illustrate how these gene fusions have been used to study the regulation and structure of transport proteins in Escherichia coli.