Bioinformatics and experimental analysis of proteins of two-component systems in Myxococcus xanthus

Abstract

Proteins of two-component systems (TCS) have essential functions in the sensing of external and self-generated signals in bacteria and in the generation of appropriate output responses. Accordingly, in Myxococcus xanthus, TCS are important for normal motility and fruiting body formation and sporulation. Here we analyzed the M. xanthus genome for the presence and genetic organization of genes encoding TCS. Two hundred seventy-two TCS genes were identified, 251 of which are not part of che gene clusters. We report that the TCS genes are unusually organized, with 55% being orphan and 16% in complex gene clusters whereas only 29% display the standard paired gene organization. Hybrid histidine protein kinases and histidine protein kinases predicted to be localized to the cytoplasm are overrepresented among proteins encoded by orphan genes or in complex gene clusters. Similarly, response regulators without output domains are overrepresented among proteins encoded by orphan genes or in complex gene clusters. The most frequently occurring output domains in response regulators are involved in DNA binding and cyclic-di-GMP metabolism. Our analyses suggest that TCS encoded by orphan genes and complex gene clusters are functionally distinct from TCS encoded by paired genes and that the connectivity of the pathways made up of TCS encoded by orphan genes and complex gene clusters is different from that of pathways involving TCS encoded by paired genes. Experimentally, we observed that orphan TCS genes are overrepresented among genes that display altered transcription during fruiting body formation. The systematic analysis of the 25 orphan genes encoding histidine protein kinases that are transcriptionally up-regulated during development showed that 2 such genes are likely essential for viability and identified 7 histidine protein kinases, including 4 not previously characterized that have important function in fruiting body formation or spore germination.

FIG. 1.

Classification scheme for two-component system genes. Schematic diagram of classification schemes for two-component system genes. The definition of paired and orphan TCS genes includes information about transcription direction as indicated by the arrow symbols. Complex TCS gene clusters include clusters containing two or more RR genes, clusters containing two or more HPK or HPK-like genes, and clusters with three or more TCS genes irrespective of transcription direction, as indicated by the box symbols. For complex gene clusters, only the most common gene organizations are shown (see Tables S3, S4, and S5 in the supplemental material for all gene organizations found in these clusters).

FIG. 2.

Outline of strategy for generating in-frame deletions in genes encoding histidine protein kinases. For each gene to be deleted, a plasmid was constructed by PCR and cloning with approximately 550-bp regions of homology upstream (light gray) or downstream (dark gray) flanking the in-frame construct. In a two-step procedure, deletion strains were isolated by first selecting for kanamycin resistance, followed by galactose counterselection using the galK gene carried on the plasmid and screening for loss of kanamycin resistance. Cells harboring galK die in the presence of galactose. Candidate clones for carrying the in-frame deletion were identified as Gal^r and Kan^S. Clones carrying the in-frame deletion were identified by PCR using the primer pairs EF and GH. For simplicity, the first homologous recombination is shown only for the recombination occurring in the upstream flanking region.

FIG. 3.

Organization diagram of histidine protein kinase genes and proteins in the M. xanthus genome. HPK genes were divided into three categories based on genetic organization. The number of hybrid HPKs is indicated in parentheses for each category. In the second layer, HPKs are divided according to their likely subcellular localization based on the presence (integral membrane) or absence (cytoplasmic) of transmembrane helices. In the third layer, HPK genes are divided according to transcriptional regulation during development. Note that in the third layer, not all HPK genes are included because they are not all present on the M. xanthus DNA microarray or they were not tested by qRT-PCR.

FIG. 4.

Organization diagram of response regulator genes and proteins in the M. xanthus genome. RR genes were divided into three categories based on genetic organization. In the second layer, RRs are divided according to the presence or absence of output domains. In the third layer, RRs are divided according to transcriptional regulation during development. Note that in the third layer, not all RR genes are included because they are not all present on the M. xanthus DNA microarray or they were not tested by qRT-PCR.

FIG. 5.

Organization diagram of histidine protein kinase-like genes and proteins in the M. xanthus genome. Genes encoding HPK-like proteins were divided into three categories based on genetic organization. In the second layer, HPK-like proteins are divided according to their likely subcellular localization based on the presence (integral membrane) or absence (cytoplasmic) of transmembrane helices. Note that in the third layer, not all HPK-like genes are included because they are not all present on the M. xanthus DNA microarray or they were not tested by qRT-PCR.

FIG. 6.

Developmental phenotypes of mutants containing in-frame deletions of orphan histidine protein kinase genes. (A) Developmental phenotypes on CF agar. The indicated strains were starved on CF agar for the indicated periods of time. All strains analyzed are derivatives of DK1622; below strain numbers, the in-frame deletion present in a particular strain is indicated. Scale bar, 1.0 mm. (B) Developmental phenotypes in submerged culture. The same strains as in panel A were exposed to starvation in submerged culture for 120 h. Scale bar, 100 μm.