Cui-ying Zhang

Characteristics and Living Patterns of Marine Myxobacterial Isolates

ABSTRACT The growth, morphology, and life cycle of two marine myxobacterial isolates, halotolerant Myxococcus fulvus strain HW-1 and halophilic Haliangium ochraceum strain SMP-2, were studied as models to determine the living patterns of myxobacteria in the ocean. The growth, morphology, and development of halotolerant strain HW-1 shifted in response to salinity. The optimal seawater concentration for growth of HW-1 was 0 to 80% (salinity, 0.1 to 2.9%), and the strain grew poorly in media with a salinity of more than 4%. The cells became shorter as the seawater concentration increased. The fruiting body structure was complete only on agar prepared with low concentrations of seawater or salts (less than 60% seawater; salinity, 2.1%), and rudimentary structures or even simple cell mounds appeared as the seawater concentration increased. In contrast, the halophilic strain SMP-2 was unable to grow without NaCl. The cell length and the morphology of the fruiting body-like structure did not change in response to salts. In seawater liquid medium, the cells of both strains were confirmed to be able to form myxospores directly from vegetative cells, but they could not do so in medium containing a low seawater concentration (10% or less). HW-1 cells from medium containing a high concentration of seawater grew independent of cell density, while cells from medium containing a low concentration of seawater (10% or less) showed density-dependent growth. SMP-2 cells showed density-dependent growth under all salinity conditions. The results suggest that the halotolerant myxobacteria are the result of degenerative adaptation of soil myxobacteria to the marine environment, while the halophilic myxobacteria form a different evolutionary group that is indigenous to the ocean.

Myxococcus xanthus Viability Depends on GroEL Supplied by Either of Two Genes, but the Paralogs Have Different Functions during Heat Shock, Predation, and Development

Myxococcus xanthus DK1622 contains two paralogous groEL gene loci that possess both different sequences and different organizations within the genome. Deletion of either one of these two genes alone does not affect cell viability. However, deletion of both groEL genes results in cell death unless a complemented groEL1 or groEL2 gene is present. The groEL1 gene was determined to be essential for cell survival under heat shock conditions; a strain with mutant groEL2 caused cells to be more sensitive than the wild-type strain to higher temperatures. Mutants with a single deletion of either groEL1 (MXAN_4895) or groEL2 (MXAN_4467) had a growth curve similar to that of the wild-type strain DK1622 in medium containing hydrolyzed proteins as the substrate. However, when cells were cultured on medium containing either Escherichia coli cells or casein as the substrate, deletion of groEL2, but not groEL1, led to a deficiency in cell predation and macromolecular feeding. Furthermore, groEL1 was found to play an indispensable role in the development and sporulation of cells, but deletion of groEL2 had no visible effects. Our results suggest that, although alternatively required for cell viability, the products of the two groEL genes have divergent functions in the multicellular social life cycle of M. xanthus DK1622.

Taxonomic analysis of Sorangium strains based on HSP60 and 16S rRNA gene sequences and morphology.

The taxonomy of myxobacteria is based mainly on their morphological characteristics. The genus Sorangium belongs to the myxobacterial suborder Sorangiineae. Strains in the genus were classified either as one species, Sorangium cellulosum, by ignoring divergent morphological characteristics, or into several species; however, the latter classification is based on some dubious morphological characteristics and is inconsistent with the phylogeny constructed from 16S rRNA gene sequences. In this study, two HSP60 (groEL1 and groEL2) genes were amplified and sequenced from 22 Sorangium strains. The groEL1 and groEL2 gene sequences were highly conserved in Sorangium strains, suggesting that these two paralogous genes both play important roles in the life cycle. The phylogeny constructed by the groEL genes was rather consistent with the morphological characteristics of sporangioles. Including information from the phylogenetic analysis and morphological characteristics, it is suggested that the genus Sorangium includes two species.

Seawater-Regulated Genes for Two-Component Systems and Outer Membrane Proteins in Myxococcus

When salt-tolerant Myxococcus cells are moved to a seawater environment, they change their growth, morphology, and developmental behavior. Outer membrane proteins and signal transduction pathways may play important roles in this shift. Chip hybridization targeting the genes predicted to encode 226 two-component signal transduction pathways and 74 outer membrane proteins of M. xanthus DK1622 revealed that the expression of 55 corresponding genes in the salt-tolerant strain M. fulvus HW-1 was significantly modified (most were downregulated) by the presence of seawater. Sequencing revealed that these seawater-regulated genes are highly homologous in both strains, suggesting that they have similar roles in the lifestyle of Myxococcus. Seven of the genes that had been reported in M. xanthus DK1622 are involved in different cellular processes, such as fruiting body development, sporulation, or motility. The outer membrane (Om) gene Om031 had the most significant change in expression (downregulated) in response to seawater, while the two-component system (Tc) gene Tc105 had the greatest increase in expression. Their homologues MXAN3106 and MXAN4042 were knocked out in DK1622 to analyze their functions in response to changes in salinity. In addition to having increased salt tolerance, sporulation of the MXAN3106 mutant was enhanced compared to that of DK1622, whereas mutating gene MXAN4042 produced contrary results. The results indicated that the genes that are involved in the cellular processes that are significantly changed in response to salinity may also be involved the salt tolerance of Myxococcus cells. Regulating the expression levels of these multifunctional genes may allow cells to quickly and efficiently respond to changing conditions in coastal environments.

Adaptation of Salt-tolerant Myxococcus Strains and their Motility Systems to the Ocean Conditions

More and more studies have indicated that myxobacteria are able to live in seawater conditions, which, however, can decrease the fruiting body formation ability and also the adventurous (A) and social (S) motility systems of the myxobacteria. To learn the adaptation mechanism of the salt-tolerant myxobacteria to marine conditions, we analyzed 10 salt-tolerant Myxococcus strains of their fruiting body formation and motility. The isolates were from marine samples and possessed different levels of salt tolerance. They had the dual motility system and formed fruiting bodies in the presence of suitable seawater concentrations. Some high salt-tolerant strains even lost their fruiting abilities in the absence of seawater. In response to the presence of seawater, the S-motility was found to be increased in the high salt-tolerants but decreased in the low salt-tolerants. The A-motility, on the other hand, was observed in all the salt-tolerant Myxococcus strains, but increased or decreased in response to the presence of seawater. Perceived shifts of fruiting body formation abilities and motilities discovered in the salt-tolerant Myxococcus strains suggested an ecological adaptation of myxobacterial social behaviors to the marine environments.

Hdsp, a horizontally transferred gene required for social behavior and halotolerance in salt-tolerant Myxococcus fulvus HW-1

Myxococcus fulvus HW-1, a salt-tolerant bacterial strain, which was isolated from a coastal environment, changes its behavior with different salinities. To study the relationship between behavioral shifts and the adaption to oceanic conditions, the HW-1 strain was randomly mutagenized using transposon insertion, producing a dispersed-growing mutant, designated YLH0401. The mutant did not develop fruiting bodies and myxospores, was deficient in S-motility, produced less extracellular matrix and was less salt tolerant. The YLH0401 strain was determined to be mutated by a single insertion in a large gene of unknown function (7011 bp in size), which is located in a horizontally transferred DNA fragment. The gene is expressed during the vegetative growth stage, as well as highly and stably expressed during the development stage. This horizontally transferred gene may allow Myxococcus to adapt to oceanic conditions.

New Locus Important for Myxococcus Social Motility and Development

The mts locus in salt-tolerant Myxococcus fulvus HW-1 was found to be critical for gliding motility, fruiting-body formation, and sporulation. The homologous genes in Myxococcus xanthus are also important for social motility and fruiting-body development. The mts genes were determined to be involved in cell-cell cohesion in both myxobacterial species.

Improving Cellular Properties for Genetic Manipulation by Dispersed Growing Mutagenesis in Myxococcus fulvus HW-1

One of the key limitations to genetic manipulation in myxobacteria is that the cells grow in clumps in liquid. A salt-tolerant strain HW-1 of Myxococcus fulvus was treated with UV irradiation and produced a completely dispersedly growing mutant UV684. There were no significant differences between the parent HW-1 and the mutant UV684 in terms of salt-tolerant growth. The mutant UV684 and the parent strain had the similar abilities of the fruiting body formation and S motility. Interestingly, the mutant exhibited high transformation/transposition efficiency with 105–106 colony-forming units per μg DNA, which was about 103–105 fold higher than HW-1. The results indicate that the mutation that led to dispersed growth in the UV684 mutant strain had a few impacts on social behavior, but greatly facilitated molecular genetic manipulation.