Refined crystal structure of beta-lactamase from Citrobacter freundii indicates a mechanism for beta-lactam hydrolysis

Beta-Lactamases (EC 3.5.2.6, 'penicillinases') are a family of enzymes that protect bacteria against the lethal effects of cell-wall synthesis of penicillins, cephalosporins and related antibiotic agents, by hydrolysing the beta-lactam antibiotics to biologically inactive compounds. Their production can, therefore, greatly contribute to the clinical problem of antibiotic resistance. Three classes of beta-lactamases--A, B and C--have been identified on the basis of their amino-acid sequence; class B beta-lactamases are metalloenzymes, and are clearly distinct from members of class A and C beta-lactamases, which both contain an active-site serine residue involved in the formation of an acyl enzyme with beta-lactam substrates during catalysis. It has been predicted that class C beta-lactamases share common structural features with D,D-carboxypeptidases and class A beta-lactamases, and further, suggested that class A and class C beta-lactamases have the same evolutionary origin as other beta-lactam target enzymes. We report here the refined three-dimensional structure of the class C beta-lactamase from Citrobacter freundii at 2.0-A resolution and confirm the predicted structural similarity. The refined structure of the acyl-enzyme formed with the monobactam inhibitor aztreonam at 2.5-A resolution defines the enzyme's active site and, along with molecular modelling, indicates a mechanism for beta-lactam hydrolysis. This leads to the hypothesis that Tyr 150 functions as a general base during catalysis.     

Nitration of a peptide phytotoxin by bacterial nitric oxide synthase

Nitric oxide (NO) is a potent intercellular signal in mammals that mediates key aspects of blood pressure, hormone release, nerve transmission and the immune response of higher organisms. Proteins homologous to full-length mammalian nitric oxide synthases (NOSs) are found in lower multicellular organisms. Recently, genome sequencing has shown that some bacteria contain genes coding for truncated NOS proteins; this is consistent with reports of NOS-like activities in bacterial extracts. Biological functions for bacterial NOSs are unknown, but have been presumed to be analogous to their role in mammals. Here we describe a gene in the plant pathogen Streptomyces turgidiscabies that encodes a NOS homologue, and we reveal its role in nitrating a dipeptide phytotoxin required for plant pathogenicity. High similarity between bacterial NOSs indicates a general function in biosynthetic nitration; thus, bacterial NOSs constitute a new class of enzymes. Here we show that the primary function of Streptomyces NOS is radically different from that of mammalian NOS. Surprisingly, mammalian NO signalling and bacterial biosynthetic nitration share an evolutionary origin.     

Tertiary structural differences between microbial serine proteases and pancreatic serine enzymes.

Although primary structural homology between bacterial serine proteases and those from the mammalian pancreas is slight, two-thirds of the residues in the bacterial enzyme SGPB as seen at 2.8-A resolution, adopt a similar polypeptide chain conformation to that of the chymotrypsin family. The three major regions of difference show how this family of proteolytic enzymes has developed from the more primitive bacterial to the relatively sophisticated pancreatic enzymes.     

Relationship of a putative receptor protein kinase from maize to the S-locus glycoproteins of Brassica

The protein kinase family of enzymes mediates the responses of eukaryotic cells to both inter- and intracellular signals. These enzymes are either serine/threonine-specific or tyrosine-specific. Many of the latter are transmembrane receptors and are important in transduction of extracellular signals across the plasma membrane, whereas few examples of receptor serine kinases have been reported. We have now identified a complementary DNA clone from Zea mays (L.) encoding a putative serine/threonine-specific protein kinase structurally related to the receptor tyrosine kinases. This structural similarity is evidence for a previously undescribed class of transmembrane receptor in higher plants likely to be involved in signal reception and transduction. Furthermore, the catalytic domain of this protein kinase is linked through a transmembrane domain to an extracellular domain similar to that of glycoproteins encoded in the self-incompatibility locus of Brassica which are involved in the self-recognition system between pollen and stigma.     

Bacterial growth and primary production along a north-south transect of the Atlantic Ocean

The oceanic carbon cycle is mainly determined by the combined activities of bacteria and phytoplankton, but the interdependence of climate, the carbon cycle and the microbes is not well understood. To elucidate this interdependence, we performed high-frequency sampling of sea water along a north-south transect of the Atlantic Ocean. Here we report that the interaction of bacteria and phytoplankton is closely related to the meridional profile of water temperature, a variable directly dependent on climate. Water temperature was positively correlated with the ratio of bacterial production to primary production, and, more strongly, with the ratio of bacterial carbon demand to primary production. In warm latitudes (25 degrees N to 30 degrees S), we observed alternating patches of predominantly heterotrophic and autotrophic community metabolism. The calculated regression lines (for data north and south of the Equator) between temperature and the ratio of bacterial production to primary production give a maximum value for this ratio of 40% in the oligotrophic equatorial regions. Taking into account a bacterial growth efficiency of 30%, the resulting area of net heterotrophy (where the bacterial carbon demand for growth plus respiration exceeds phytoplankton carbon fixation) expands from 8 degrees N (27 degrees C) to 20 degrees S (23 degrees C). This suggests an output of CO2 from parts of the ocean to the atmosphere.     

Crystal structure of a self-splicing group I intron with both exons

The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. Here we report the X-ray crystal structure (3.1-A resolution) of a complete group I bacterial intron in complex with both the 5'- and the 3'-exons. This complex corresponds to the splicing intermediate before the exon ligation step. It reveals how the intron uses structurally unprecedented RNA motifs to select the 5'- and 3'-splice sites. The 5'-exon's 3'-OH is positioned for inline nucleophilic attack on the conformationally constrained scissile phosphate at the intron-3'-exon junction. Six phosphates from three disparate RNA strands converge to coordinate two metal ions that are asymmetrically positioned on opposing sides of the reactive phosphate. This structure represents the first splicing complex to include a complete intron, both exons and an organized active site occupied with metal ions.     

Robustness-epistasis link shapes the fitness landscape of a randomly drifting protein

The distribution of fitness effects of protein mutations is still unknown. Of particular interest is whether accumulating deleterious mutations interact, and how the resulting epistatic effects shape the protein's fitness landscape. Here we apply a model system in which bacterial fitness correlates with the enzymatic activity of TEM-1 beta-lactamase (antibiotic degradation). Subjecting TEM-1 to random mutational drift and purifying selection (to purge deleterious mutations) produced changes in its fitness landscape indicative of negative epistasis; that is, the combined deleterious effects of mutations were, on average, larger than expected from the multiplication of their individual effects. As observed in computational systems, negative epistasis was tightly associated with higher tolerance to mutations (robustness). Thus, under a low selection pressure, a large fraction of mutations was initially tolerated (high robustness), but as mutations accumulated, their fitness toll increased, resulting in the observed negative epistasis. These findings, supported by FoldX stability computations of the mutational effects, prompt a new model in which the mutational robustness (or neutrality) observed in proteins, and other biological systems, is due primarily to a stability margin, or threshold, that buffers the deleterious physico-chemical effects of mutations on fitness. Threshold robustness is inherently epistatic-once the stability threshold is exhausted, the deleterious effects of mutations become fully pronounced, thereby making proteins far less robust than generally assumed.     

Expression of a transposable antibiotic resistance element in Saccharomyces

Some eukaryotic genes can be expressed in bacteria but there are few examples of the expression of prokaryotic genes in eukaryotes. Antibiotic G418 is a 2-deoxystreptamine antibiotic that is structurally related to gentamicin but has inhibitory activity against a much wider variety of pro- and eukaryotic organisms. In bacteria, resistance to G418 can be determined by several plasmid-encoded modifiying enzymes and, in view of the broad spectrum of activity of G418, we considered that this antibiotic might be useful as a selective agent for the introduction of these antibiotic resistance genes into a eukaryotic organism such as Saccharomyces cerevisiae. Additional impetus for these experiments came from the knowledge that certain of the G418-resistance determinants in bacteria are carried on transposable elements; a study of the properties of these elements in eukaryotes would be intriguing.     

Urea excretion as a strategy for survival in a fish living in a very alkaline environment

Ammonia is toxic to all vertebrates. It can be converted to the less toxic urea, but this is a metabolically expensive process found only in terrestrial vertebrates that cannot readily excrete ammonia and marine fish that use urea as an osmotic filler. Freshwater fish mostly excrete ammonia with only a small quantity of urea. It seems the ornithine cycle for urea production has been suppressed in all freshwater teleosts except for some airbreathers which, when exposed to air, increase urea synthesis via the cycle. Here we show that the tilapia fish Oreochromis alcalicus grahami, the only fish living in Lake Magadi, an alkaline soda lake (pH = 9.6-10) in the Kenyan Rift Valley, excretes exclusively urea and has ornithine-urea cycle enzymes in its liver. A closely related species that lives in water at pH 7.1 lacks these enzymes and excretes mainly ammonia with small amounts of urea produced via uricolysis. It dies within 60 min when placed in water from Lake Magadi. We suggest that urea production via the ornithine-urea cycle permits O. a. grahami to survive the very alkaline conditions in Lake Magadi.     

Blocker protection in the pore of a voltage-gated K+ channel and its structural implications

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow 'inner pore' seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.     

The assembly of a GTPase-kinase signalling complex by a bacterial catalytic scaffold

The fidelity and specificity of information flow within a cell is controlled by scaffolding proteins that assemble and link enzymes into signalling circuits. These circuits can be inhibited by bacterial effector proteins that post-translationally modify individual pathway components. However, there is emerging evidence that pathogens directly organize higher-order signalling networks through enzyme scaffolding, and the identity of the effectors and their mechanisms of action are poorly understood. Here we identify the enterohaemorrhagic Escherichia coli O157:H7 type III effector EspG as a regulator of endomembrane trafficking using a functional screen, and report ADP-ribosylation factor (ARF) GTPases and p21-activated kinases (PAKs) as its relevant host substrates. The 2.5?? crystal structure of EspG in complex with ARF6 shows how EspG blocks GTPase-activating-protein-assisted GTP hydrolysis, revealing a potent mechanism of GTPase signalling inhibition at organelle membranes. In addition, the 2.8?? crystal structure of EspG in complex with the autoinhibitory Iα3-helix of PAK2 defines a previously unknown catalytic site in EspG and provides an allosteric mechanism of kinase activation by a bacterial effector. Unexpectedly, ARF and PAKs are organized on adjacent surfaces of EspG, indicating its role as a 'catalytic scaffold' that effectively reprograms cellular events through the functional assembly of GTPase-kinase signalling complex.     

Legionella pneumophila SidD is a deAMPylase that modifies Rab1

Legionella pneumophila actively modulates host vesicle trafficking pathways to facilitate its intracellular replication with effectors translocated by the Dot/Icm type IV secretion system (T4SS). The SidM/DrrA protein functions by locking the small GTPase Rab1 into an active form by its guanine nucleotide exchange factor (GEF) and AMPylation activity. Here we demonstrate that the L. pneumophila protein SidD preferably deAMPylates Rab1. We found that the deAMPylation activity of SidD could suppress the toxicity of SidM to yeast and is required to release Rab1 from bacterial phagosomes efficiently. A molecular mechanism for the temporal control of Rab1 activity in different phases of L. pneumophila infection is thus established. These observations indicate that AMPylation-mediated signal transduction is a reversible process regulated by specific enzymes.     

黑臭河道底泥细菌群落结构对人工曝气响应的DGGE分析

采用基于16S rDNA的PCR-DGGE(变性梯度凝胶电泳)图谱并通过条带割胶回收DNA进行序列分析,初步探讨了不同曝气条件下黑臭河道底泥中细菌群落结构多样性及其变化,同时用冗余分析(RDA)研究了环境因子与细菌群落结构之间的关系.结果表明,人工曝气对黑臭河道底泥细菌群落结构产生明显的影响,并且随不同曝气强度底泥细菌群落多样性呈现不同的变化,其中当曝气扰动雷诺数(Re)为1810,溶解氧(DO)为7.35时,细菌优势群落多样性最高;序列比对分析推测适度的曝气有利于促进碳、氮、硫循环相关细菌的生长,其中以变形菌门为主导;冗余分析显示DO和Re对细菌群落结构影响显著.     

地毯草黄单胞菌ABB-2对红掌防御相关生化指标的影响

研究了地毯草黄单胞菌（Xanthomonas axonopodis）ABB-2对红掌主栽品种阿拉巴马的致病性及其防御相关生化指标的动态影响．结果表明，红掌阿拉巴马对该病菌高度敏感，离体叶片接种第4d、盆栽植株接种第8d后，被接种叶片的病叶率达到100%．红掌接种该病菌后，体内超氧化物歧化酶（SOD）、过氧化物酶（POD）、多酚氧化酶（PPO）和苯丙氨酸解氨酶（PAL）活性增加，分别在接种后的第4、6、8和12d达到峰值,较非接种分别对照增加了15.37、35.63、51.89和66.43%；总可溶性蛋白、游离酚含量最大值分别为非接种对照的121.76和134.66%．     

SOS response promotes horizontal dissemination of antibiotic resistance genes

Mobile genetic elements have a crucial role in spreading antibiotic resistance genes among bacterial populations. Environmental and genetic factors that regulate conjugative transfer of antibiotic resistance genes in bacterial populations are largely unknown. Integrating conjugative elements (ICEs) are a diverse group of mobile elements that are transferred by means of cell-cell contact and integrate into the chromosome of the new host. SXT is a approximately 100-kilobase ICE derived from Vibrio cholerae that encodes genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin. SXT-related elements were not detected in V. cholerae before 1993 but are now present in almost all clinical V. cholerae isolates from Asia. ICEs related to SXT are also present in several other bacterial species and encode a variety of antibiotic and heavy metal resistance genes. Here we show that SetR, an SXT encoded repressor, represses the expression of activators of SXT transfer. The 'SOS response' to DNA damage alleviates this repression, increasing the expression of genes necessary for SXT transfer and hence the frequency of transfer. SOS is induced by a variety of environmental factors and antibiotics, for example ciprofloxacin, and we show that ciprofloxacin induces SXT transfer as well. Thus, we present a mechanism by which therapeutic agents can promote the spread of antibiotic resistance genes.     

Fine-scale phylogenetic architecture of a complex bacterial community

Although molecular data have revealed the vast scope of microbial diversity, two fundamental questions remain unanswered even for well-defined natural microbial communities: how many bacterial types co-exist, and are such types naturally organized into phylogenetically discrete units of potential ecological significance? It has been argued that without such information, the environmental function, population biology and biogeography of microorganisms cannot be rigorously explored. Here we address these questions by comprehensive sampling of two large 16S ribosomal RNA clone libraries from a coastal bacterioplankton community. We show that compensation for artefacts generated by common library construction techniques reveals fine-scale patterns of community composition. At least 516 ribotypes (unique rRNA sequences) were detected in the sample and, by statistical extrapolation, at least 1,633 co-existing ribotypes in the sampled population. More than 50% of the ribotypes fall into discrete clusters containing less than 1% sequence divergence. This pattern cannot be accounted for by interoperon variation, indicating a large predominance of closely related taxa in this community. We propose that such microdiverse clusters arise by selective sweeps and persist because competitive mechanisms are too weak to purge diversity from within them.     

Effect of pH values on the extracellular polysaccharide secreted by Acidithiobacillus ferrooxidans during chalcopyrite bioleaching

The pH value plays an important role in the bioleaching of sulphide minerals. The effect of pH values on the extracellular poly-saccharide secreted by Acidithiobacillus ferrooxidans was investigated in different phases of bacterial growth during chalcopyrite bioleach-ing. It is found that extracellular polysaccharide secretion from the cells attached to chalcopyrite is more efficiently than that of the free cells in the bioleaching solution. Three factors, pH values, the concentration of soluble metal ions, and the bacterial growth and metabolism, affect extracellular polysaccharide secretion in the free cells, and are related to the bacterial growth phase. Extracellular polysaccharide secretion from the attached cells is mainly dependent on the pH value of the bacterial culture.     

Chloroquine diverts ACTH from a regulated to a constitutive secretory pathway in AtT-20 cells

AtT-20 cells, a mouse pituitary line, externalize a viral membrane glycoprotein and the precursor of ACTH constitutively, that is, rapidly without storage or regulation. They also have a regulated pathway in which they cleave the precursor to mature hormones, ACTH and beta-endorphin, store them in secretory granules and discharge them only in the presence of a secretagogue. An analogy exists for newly synthesized lysosomal enzymes which are either delivered to the lysosome or secreted from the cell. Targeting to the lysosomes may require a low pH step, since chloroquine causes the enzymes to be secreted from the cell. Here we show that chloroquine (200 microM) also appears to block the storage of newly synthesized ACTH in secretory granules and instead diverts it to the outside of the cell via the constitutive pathway. Chloroquine has no effect on the constitutive pathway and does not block the exocytosis of pre-packaged ACTH. Thus like lysosomal enzymes, peptide hormones are not sent to their correct destinations in the presence of chloroquine, but are diverted instead to a constitutive pathway that is chloroquine-insensitive.