The TatC component of the twin‐arginine protein translocase functions as an obligate oligomer

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate‐bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild‐type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue‐native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild‐type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate‐induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.     

The twin arginine protein transport pathway exports multiple virulence proteins in the plant pathogen Streptomyces scabies

Streptomyces scabies is one of a group of organisms that causes the economically important disease potato scab. Analysis of the S. scabies genome sequence indicates that it is likely to secrete many proteins via the twin arginine protein transport (Tat) pathway, including several proteins whose coding sequences may have been acquired through horizontal gene transfer and share a common ancestor with proteins in other plant pathogens. Inactivation of the S. scabies Tat pathway resulted in pleiotropic phenotypes including slower growth rate and increased permeability of the cell envelope. Comparison of the extracellular proteome of the wild type and ΔtatC strains identified 73 predicted secretory proteins that were present in reduced amounts in the tatC mutant strain, and 47 Tat substrates were verified using a Tat reporter assay. The ΔtatC strain was almost completely avirulent on Arabidopsis seedlings and was delayed in attaching to the root tip relative to the wild‐type strain. Genes encoding 14 candidate Tat substrates were individually inactivated, and seven of these mutants were reduced in virulence compared with the wild‐type strain. We conclude that the Tat pathway secretes multiple proteins that are required for full virulence.     

Identification and molecular characterization of twin-arginine translocation system (Tat) in <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> strain PXO99

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, one of the most widespread and destructive bacterial diseases in rice. This study identified and characterized the contribution of the twin-arginine translocation (Tat) pathway to motility, chemotaxis, extracellular polysaccharide (EPS) production and virulence in X. oryzae pv. oryzae strain PXO99. The tatC disruption mutant (strain TCM) of strain PXO99 were generated, and confirmed both by PCR and Southern blotting. Strain PXO99 cells were highly motile in NYGB 0.3% soft agar plate. In contrast, the tatC mutation impaired motility. Furthermore, strain TCM cells lacked detectable flagella and exhibited almost no chemotaxis toward glucose under aerobic conditions, indicating that the Tat secretion pathway contributed to flagellar biogenesis and chemotactic responses. It was also observed that strain TCM exhibited a reductive production of extracellular polysaccharide (EPS) and a significant reduction of virulence on rice plants when compared with the wild type PXO99. However, the tatC mutation in strain PXO99 did not affect growth rate and the ability to induce hypersensitive response (HR) in nonhost tobacco (Nicotiana tabacum L. cv. Samsun). Our findings indicated that the Tat system of X. oryzae pv. oryzae played an important role in the pathogen’s virulence. L. Chen, B. Hu, and G. Qian contributed equally to this research.     

Efficient export of prefolded,disulfide‐bonded recombinant proteins to the periplasm by the Tat pathway in Escherichia coli CyDisCo strains

Numerous high‐value therapeutic proteins are produced in Escherichia coli and exported to the periplasm, as this approach simplifies downstream processing and enables disulfide bond formation. Most recombinant proteins are exported by the Sec pathway, which transports substrates across the plasma membrane in an unfolded state. The Tat system also exports proteins to the periplasm, but transports them in a folded state. This system has attracted interest because of its tendency to transport correctly folded proteins, but this trait renders it unable to export proteins containing disulfide bonds since these are normally acquired only in the periplasm; reduced substrates tend to be recognized as incorrectly folded and rejected. In this study we have used a series of novel strains (termed CyDisCo) which oxidise disulfide bonds in the cytoplasm, and we show that these cells efficiently export a range of disulfide‐containing proteins when a Tat signal peptide is attached. These test proteins include alkaline phosphatase (PhoA), a phytase containing four disulfide bonds (AppA), an antiinterleukin 1β scFv and human growth hormone. No export of PhoA or AppA is observed in wild‐type cells lacking the CyDisCo factors. The PhoA, AppA and scFv proteins were exported in an active form by Tat in the CyDisCo strain, and mass spectrometry showed that the vast majority of the scFv protein was disulfide‐bonded and correctly processed. The evidence indicates that this combination of Tat + CyDisCo offers a novel means of exporting active, correctly folded disulfide bonded proteins to the periplasm. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:281–290, 2014     

Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins

The twin-arginine translocation (Tat) pathway is a system used by some bacteria to export proteins out from the cytosol to the cell surface or extracellular environment. A functional Tat pathway exists in the important human pathogen Mycobacterium tuberculosis. Identification of the substrates exported by the Tat pathway can help define the role that this pathway plays in the physiology and pathogenesis of M. tuberculosis. Here we used a reporter of Tat export, a truncated β-lactamase, ′BlaC, to experimentally identify M. tuberculosis proteins with functional Tat signal sequences. Of the 13 proteins identified, one lacks the hallmark of a Tat-exported substrate, the twin-arginine dipeptide, and another is not predicted by in silico analysis of the annotated M. tuberculosis genome. Full-length versions of a subset of these proteins were tested to determine if the native proteins are Tat exported. For three proteins, expression in a Δtat mutant of Mycobacterium smegmatis revealed a defect in precursor processing compared to expression in the wild type, indicating Tat export of the full-length proteins. Conversely, two proteins showed no obvious Tat export in M. smegmatis. One of this latter group of proteins was the M. tuberculosis virulence factor phospholipase C (PlcB). Importantly, when tested in M. tuberculosis a different result was obtained and PlcB was exported in a twin-arginine-dependent manner. This suggests the existence of an M. tuberculosis-specific factor(s) for Tat export of a proven virulence protein. It also emphasizes the importance of domains beyond the Tat signal sequence and bacterium-specific factors in determining if a given protein is Tat exported.     

Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.     

TatD is a central component of a Tat translocon‐initiated quality control system for exported FeS proteins in Escherichia coli

Bacterial Tat systems export folded proteins, including FeS proteins such as NrfC and NapG, which acquire their cofactors before translocation. NrfC and NapG are proofread by the Tat pathway, and misfolded examples are degraded after interaction with the translocon. Here, we identify TatD as a crucial component of this quality control system in Escherichia coli. NrfC/NapG variants lacking FeS centres are rapidly degraded in wild‐type cells but stable in a ΔtatD strain. The precursor of another substrate, FhuD, is also transiently detected in wild‐type cells but stable in the ΔtatD strain. Surprisingly, these substrates are stable in ΔtatD cells that overexpress TatD, and export of the non‐mutated precursors is inhibited. We propose that TatD is part of a quality control system that is intimately linked to the Tat export pathway, and that the overexpression of TatD leads to an imbalance between the two systems such that both Tat‐initiated turnover and export are prevented.     

Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery

The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild‐type Tat system exporting the over‐expressed substrate protein FhuD. These were compared to strains cell‐engineered to over‐express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over‐expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over‐expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild‐type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5 mg L^?1 of growth medium. Cells over‐expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40‐fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over‐expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method—a scaleable way to release periplasmic protein—showed similar robustness between strains. A scale‐down mimic of continuous disk‐stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over‐expressing the Tat pathway performed comparably to cells with the wild‐type system. Overall, engineering E. coli cells to over‐express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance. Biotechnol. Bioeng. 2012; 109:983–991. © 2011 Wiley Periodicals, Inc.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein,TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

Background

The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence.

Methodology/Principal Findings

The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species.

Conclusions/Significance

TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host.     

A genetic analysis of in vivo selenate reduction by <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium LT2 and <Emphasis Type="Italic">Escherichia coli</Emphasis> K12

The twin-arginine transport (Tat) system is dedicated to the translocation of folded proteins across the bacterial cytoplasmic membrane. Proteins are targeted to the Tat system by signal peptides containing a twin-arginine motif. In Salmonella enterica serovar Typhimurium and Escherichia coli many Tat substrates are known or predicted to bind a molybdenum cofactor in the cytoplasm prior to export. In the case of N- and S-oxide reductases, co-ordination of molybdenum cofactor insertion with protein export involves a ‘Tat proofreading’ process where chaperones of the TorD family bind the signal peptides, thus preventing premature export. Here, a genetic approach was taken to determine factors required for selenate reductase activity in Salmonella and E. coli. It is reported for both biological systems that an active Tat translocase and a TorD-like chaperone (DmsD) are required for complete in vivo reduction of selenate to elemental red selenium. Further mutagenesis and in vitro biophysical experiments implicate the Salmonella ynfE gene product, and the E. coli YnfE and YnfF proteins, as putative Tat-targeted selenate reductases.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli

Numerous high‐value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin‐arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed‐batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial‐type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA‐GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co‐expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over‐expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed‐batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h^?1, the cultures reached OD₆₀₀ values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L^?1 culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec‐dependent export approaches. Biotechnol. Bioeng. 2012; 109: 2533–2542. © 2012 Wiley Periodicals, Inc.     

Bacterial Community Dynamics During the Application of a Myxococcus xanthus-Inoculated Culture Medium Used for Consolidation of Ornamental Limestone

In this study, we investigated under laboratory conditions the bacterial communities inhabiting quarry and decayed ornamental carbonate stones before and after the application of a Myxococcus xanthus-inoculated culture medium used for consolidation of the stones. The dynamics of the community structure and the prevalence of the inoculated bacterium, M. xanthus, were monitored during the time course of the consolidation treatment (30 days). For this purpose, we selected a molecular strategy combining fingerprinting by denaturing gradient gel electrophoresis (DGGE) with the screening of eubacterial 16S rDNA clone libraries by DGGE and sequencing. Quantification of the inoculated strain was performed by quantitative real-time PCR (qPCR) using M. xanthus-specific primers designed in this work. Results derived from DGGE and sequencing analysis showed that, irrespective of the origin of the stone, the same carbonatogenic microorganisms were activated by the application of a M. xanthus culture. Those microorganisms were Pseudomonas sp., Bacillus sp., and Brevibacillus sp. The monitoring of M. xanthus in the culture media of treated stones during the time course experiment showed disparate results depending on the applied technique. By culture-dependent methods, the detection of this bacterium was only possible in the first day of the treatment, showing the limitation of these conventional techniques. By PCR-DGGE analysis, M. xanthus was detected during the first 3–6 days of the experiment. At this time, the population of this bacterium in the culture media varied between 10⁸–10⁶ cells ml⁻¹, as showed by qPCR analyses. Thereafter, DGGE analyses showed to be not suitable for the detection of M. xanthus in a mixed culture. Nevertheless, qPCR analysis using specific primers for M. xanthus showed to be a more sensitive technique for the detection of this bacterium, revealing a population of 10⁴ cells ml⁻¹ in the culture media of both treated stones at the end of the consolidation treatment. The molecular strategy used in this study is proposed as an effective monitoring system to evaluate the impact of the application of a bacterially induced carbonate mineralization as restoration/conservation treatment for ornamental stones.     

Effects of exopolysaccharide production on liquid vegetative growth,stress survival,and stationary phase recovery in <Emphasis Type="Italic">Myxococcus xanthus</Emphasis>

Exopolysaccharide (EPS) of Myxococcus xanthus is a well-regulated cell surface component. In addition to its known functions for social motility and fruiting body formation on solid surfaces, EPS has also been proposed to play a role in multi-cellular clumping in liquid medium, though this phenomenon has not been well studied. In this report, we confirmed that M. xanthus clumps formed in liquid were correlated with EPS levels and demonstrated that the EPS encased cell clumps exhibited biofilm-like structures. The clumps protected the cells at physiologically relevant EPS concentrations, while cells lacking EPS exhibited significant reduction in long-term viability and resistance to stressful conditions. However, excess EPS production was counterproductive to vegetative growth and viable cell recovery declined in extended late stationary phase as cells became trapped in the matrix of clumps. Therefore, optimal EPS production by M. xanthus is important for normal physiological functions in liquid.     

Chemotaxis plays a role in the social behaviour of Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development. The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development. However, chemotaxis has never been clearly demonstrated in Myxococcus. In this study, we showed that M. xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients. M. xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO. Responses to attractants and repellents were additive. Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein. Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium. In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge. FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media. An frzf mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions. These observations suggest that M. xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.     

Genetics of gliding motility and development inMyxococcus xanthus

Successful development in multicellular eukaryotes requires cell-cell communication and the coordinated spatial and temporal movements of cells. The complex array of networks required to bring eukaryotic development to fruition can be modeled by the development of the simpler prokaryoteMyxococcus xanthus. As part of its life cycle,M. xanthus forms multicellular fruiting bodies containing differentiated cells. Analysis of the genes essential forM. xanthus development is possible because strains with mutations that block development can be maintained in the vegetative state. Development inM. xanthus is induced by starvation, and early events in development suggest that signaling, stages have evolved to monitor the metabolic state of the developing cell. In the absence of these signals, which include amino acids, α-keto acids, and other intermediary metabolites, the ability of cells to differentiate into myxospores is impaired. Mutations that block genes controlling gliding, motility disrupt the morphogenesis of fruiting bodies and sporogenesis in surprising ways. In this review, we present data that encourage future genetic and biochemical studies of the relationships between motility, cell-cell signaling, and development inM. xanthus.     

The Myxococcus xanthus developmental program can be delayed by inhibition of DNA replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB^A116V, was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37°C. We demonstrated that at the nonpermissive temperature the DnaB^A116V mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB^A116V mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.     

Silver Sorption to Myxococcus xanthus Biomass

This paper deals with silver sorption to Myxococcus xanthus biomass. The dry biomass of this microorganism is shown to be a good sorbent for the recovery of silver present at low solution concentrations. Between initial silver concentrations of 2 and 0.05 mM, the percentage of accumulation ranges from 8.12% to 75% of the total silver present in the solution. Transmission electron microscopy study of M. xanthus wet biomass after silver accumulation shows the sorption within the extracellular polysaccharide, on the cell wall, and in the cytoplasm. The presence of silver deposits in the cytoplasm indicates that at least two mechanisms are involved in silver sorption by this bacterium biomass. First, silver was bound to the cell surface and extracellular polysaccharide, and second, a silver intracellular deposition process took place. The higher amount of silver deposits in the extracellular polysaccharide, present abundantly in M. xanthus cells, explains the capacity of this bacterium to bind silver efficiently. The results obtained indicate that the removal of silver by M. xanthus from the diluted solutions could be used in recycling this valuable metal. One interesting observation of this investigation is the crystalline form, possibly as chlorargyrite, in which the silver deposits are found in the M. xanthus cells.