- Volume 158, Issue 8, 2012

Sialic acids are nine-carbon amino sugars that are present on all mucous membranes and are often used by bacteria as nutrients. In pathogenic Vibrio the genes for sialic acid catabolism (SAC) are known to be important for host colonization, yet the route for sialic acid uptake is not proven. Vibrio cholerae contains a tripartite ATP-independent periplasmic (TRAP) transporter, SiaPQM (VC1777–VC1779), encoded by genes within the Vibrio pathogenicity island-2 (VPI-2), which are adjacent to the SAC genes nanA, nanE and nanK. We demonstrate a correlation of the occurrence of VPI-2 and the ability of Vibrio to grow on the common sialic acid N-acetylneuraminic acid (Neu5Ac), and that a V. cholerae N16961 mutant defective in vc1777, encoding the large membrane protein component of the TRAP transporter, SiaM, is unable to grow on Neu5Ac as the sole carbon source. Using the genome context and known structures of the SiaP protein component of the TRAP transporter, we define a subfamily of Neu5Ac-specific TRAP transporters, of which the vc1777–vc1779 genes are the only representatives in V. cholerae. A recent report has suggested that an entirely different TRAP transporter (VC1927–VC1929) is the Neu5Ac transporter in V. cholerae. Bioinformatics and genomic analysis suggest strongly that this is a C4-dicarboxylate-specific TRAP transporter, and indeed disruption of vc1929 results in a defect in growth on C4-dicarboxylates but not Neu5Ac. Together these data demonstrate unequivocally that the siaPQM-encoded TRAP transporter within VPI-2 is the sole sialic acid transporter in V. cholerae.

The genome of the industrially important fungus Aspergillus niger encodes a large number of glycoside hydrolase family 18 members annotated as chitinases. We identified one of these putative chitinases, CfcI, as a representative of a distinct phylogenetic clade of homologous enzymes conserved in all sequenced Aspergillus species. Where the catalytic domain of more distantly related chitinases consists of a triosephosphate isomerase barrel in which a small additional (α+β) domain is inserted, CfcI-like proteins were found to have, in addition, a carbohydrate-binding module (CBM18) that is inserted in the (α+β) domain next to the substrate-binding cleft. This unusual domain structure and sequence dissimilarity to previously characterized chitinases suggest that CfcI has a novel activity or function different from chitinases investigated so far. Following its heterologous expression and purification, its biochemical characterization showed that CfcI displays optimal activity at pH 4 and 55–65 °C and degrades chitin oligosaccharides by releasing N-acetylglucosamine from the reducing end, possibly via a processive mechanism. This is the first fungal family 18 exochitinase described, to our knowledge, that exclusively releases monomers. The cfcI expression profile suggests that its physiological function is important in processes that take place during the late stages of the aspergillus life cycle, such as autolysis or sporulation.

The Sinorhizobium meliloti locus necessary for the utilization of erythritol as a sole carbon source, contains 17 genes, including genes that encode an ABC transporter necessary for the transport of erythritol, as well as the genes encoding EryA, EryB, EryC, TpiB and the regulators EryD and EryR (SMc01615). Construction of defined deletions and complementation experiments show that the other genes at this locus encode products that are necessary for the catabolism of adonitol (ribitol) and l-arabitol, but not d-arabitol. These analyses show that aside from one gene that is specific for the catabolism of l-arabitol (SMc01619, lalA), the rest of the catabolic genes are necessary for both polyols (SMc01617, rbtC; SMc01618, rbtB; SMc01622, rbtA). Genetic and biochemical data show that in addition to utilizing erythritol as a substrate, EryA is also capable of utilizing adonitol and l-arabitol. Similarly, transport experiments using labelled erythritol show that adonitol, l-arabitol and erythritol share a common transporter (MptABCDE). Quantitative RT-PCR experiments show that transcripts containing genes necessary for adonitol and l-arabitol utilization are induced by these sugars in an eryA-dependent manner.

The lipolytic enzyme family VIII currently includes only seven members but represents a group of lipolytic enzymes with interesting properties. Recently, we identified a gene encoding the family VIII lipase LipBL from the halophilic bacterium Marinobacter lipolyticus. This enzyme, like most lipolytic enzymes from family VIII, possesses two possible nucleophilic serines located in an S-X-X-K β-lactamase motif and a G-X-S-X-G lipase motif. The serine in the S-X-X-K motif is a catalytic residue, but the role of serine within the common lipase consensus sequence G-X-S-X-G has not yet been systematically studied. Here, the previously reported time-intensive procedure for purification of recombinant LipBL was replaced by one-step metal-affinity chromatography purification in the presence of ATP. Heterologous co-expression of His6-tagged LipBL with the cytoplasmic molecular chaperones GroEL/GroES was necessary to obtain catalytically active LipBL. Site-directed mutagenesis performed to map the active site of LipBL revealed that mutation of serine and lysine in the β-lactamase motif (S72-M-T-K75) to alanine abolished the enzyme activity of LipBL, in contrast to mutation of the serine in the lipase consensus motif (S321A). Furthermore, mutagenesis was performed to understand the role of the G-X-S-X-G motif and other amino acids that are conserved among family VIII esterases. We describe how mutations in the conserved G-X-S-X-G motif altered the biochemical properties and substrate specificity of LipBL. Molecular modelling results indicate the location of the G-X-S321-X-G motif in a loop close to the catalytic centre of LipBL, presumably representing a substrate-binding site of LipBL.

Hydrogenases play many roles in bacterial physiology, and use of H2 by the uptake-type enzymes of animal pathogens is of particular interest. Hydrogenases have never been studied in the pathogen Shigella, so targeted mutant strains were individually generated in the two Shigella flexneri H2-uptake enzymes (Hya and Hyb) and in the H2-evolving enzyme (Hyc) to address their roles. Under anaerobic fermentative conditions, a Hya mutant strain (hya) was unable to oxidize H2, while a Hyb mutant strain oxidized H2 like the wild-type. A hyc strain oxidized more exogenously added hydrogen than the parent. Fluorescence ratio imaging with dye JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) showed that the parent strain generated a membrane potential 15 times greater than hya. The hya mutant was also by far the most acid-sensitive strain, being even more acid-sensitive than a mutant strain in the known acid-combating glutamate-dependent acid-resistance pathway (GDAR pathway). In severe acid-challenge experiments, the addition of glutamate to hya restored survivability, and this ability was attributed in part to the GDAR system (removes intracellular protons) by mutant strain (e.g. hya/gadBC double mutant) analyses. However, mutant strain phenotypes indicated that a larger portion of the glutamate-rescued acid tolerance was independent of GadBC. The acid tolerance of the hya strains was aided by adding chloride ions to the growth medium. The whole-cell Hya enzyme became more active upon acid exposure (20 min), based on assays of hyc. Indeed, the very high rates of Shigella H2 oxidation by Hya in acid can supply each cell with 2.4×108 protons min−1. Electrons generated from Hya-mediated H2 oxidation at the inner membrane likely counteract cytoplasmic positive charge stress, while abundant proton pools deposited periplasmically likely repel proton influx during severe acid stress.

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C–A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.